Sarutobi Ayame is a female ninja with long navy blue hair tied back, always wearing red-rimmed glasses. She is a beauty with a neat yet mature impression, often maintaining a calm expression. She has a slender height and a well-balanced physique, typically active in her black ninja attire. Depending on the situation, she is highly skilled in disguise and can infiltrate various locations. However, she suffers from severe myopia; without her glasses, her vision is so poor that she cannot properly distinguish even those close to her. Normally, she has a calm, polite, and highly responsible personality. When performing missions, she prioritizes reason over emotion and displays a professional side by judging situations coldly. She has excellent patience and does not give up easily even in difficult circumstances, possessing a strong will to complete her assigned tasks. On the other hand, in daily life, she sometimes flusters those around her with her unique way of thinking and eccentric behavior. She has a rich imagination and often gets lost in her own world, and she is very honest in expressing her emotions. Sacchan is a former member of the Oniwabanshu, the Shogunate's elite ninja group, and is a skilled practitioner of ninja techniques. She is proficient in stealth and infiltration, as well as disguise, reconnaissance, and information gathering, excelling in moving undetected. Using her high physical ability and agility, she freely scales buildings and walls, and can close the distance instantly to subdue opponents. She also freely handles concealed weapons like kunai and shuriken, and is skilled in attacking and moving using wires. During combat, she is the type to establish strategies suited to the situation based on quick judgment and rich experience, showing particular strength in close quarters and surprise attacks. Sacchan's greatest characteristic is her mental strength, which does not easily waver even in extreme situations. She maintains her composure while performing dangerous missions and responds quickly to unexpected variables. Meanwhile, her severe myopia is a weakness that significantly reduces her sense of direction and field of vision if she loses her glasses, a setting often used as a comedic element in the series. While she usually provides laughs with her somewhat eccentric and unique behavior, in decisive moments, she demonstrates her skill and responsibility as a ninja, taking the lead to protect her comrades.

Tsukuyo is a female warrior characterized by her long blonde hair tied in a high ponytail. Her bangs are long enough to slightly cover one eye, and a red decorative cord is tied through her hair. She has bluish-gray eyes and a cold, sharp impression. On the left side of her face, there are deep scars from wounds received in her childhood, which she lives with openly without hiding. She usually wears a red sash over a black and navy blue kimono and carries several kunai tucked into her waist. Her lean attire and disciplined physique reflect her long combat experience. Tsukuyo has a very serious and taciturn personality, rarely speaking unnecessary words. She always judges situations calmly and tends to prioritize reason over emotion. She has a very strong sense of responsibility and a firm will to complete her assigned tasks. She does not open her heart to others easily, but once she trusts someone, she shows deep loyalty and fulfills her responsibilities to the end. Furthermore, she possesses an upright character, knowing how to admit her mistakes and never treating the weak poorly. While she is usually strict and blunt, she unexpectedly reveals a clumsy and human side in front of familiar people. Scenes where she becomes more expressive than usual when drunk also frequently appear in the work. Tsukuyo is the leader of the Hyakku, the self-defense force of Yoshiwara, and is a top-tier warrior and kunoichi with excellent combat abilities. She possesses great agility and reflexes, using a combat style that accurately targets the enemy's openings to subdue them instantly. In particular, her technique of freely throwing multiple kunai is her representative fighting style, exerting powerful force at both close and long ranges. Her situational judgment and tactical sense are also excellent, allowing her to calmly command the battlefield even when facing multiple enemies. Her physical abilities are highly disciplined due to long practical experience, and she possesses high jumping power, balance, and quick reflexes. Her physical and mental strength are also robust, often showing her continuing to fight until the end even after sustaining major injuries. Tsukuyo cherishes Yoshiwara and its people more than anyone else, possessing a strong sense of duty that prioritizes the peace of the residents over her own safety. She is a strict leader to the Hyakku members but also cares for them like family. The scars on her face are like a symbol to ensure she never forgets her past, and she accepts them proudly without shame. While she does not reveal her emotions easily, she harbors warm consideration and a strong sense of justice in her heart. With her cool judgment, outstanding skill, and firm conviction to protect the weak, Tsukuyo is a representative female character of Gintama, loved by many as a figure who possesses both strength and humanity.