Schoolteachers in Japan frequently suffer from mental health problems, resulting in a high rate of leaves of absence from work. Considering the poor condition of Japanese teachers, this study aimed to develop a questionnaire to assess self-esteem in the teaching profession for students in teacher training courses at university. As self-esteem is generally associated with health and adaptation, improving teachers’ working conditions and enhancing their self-esteem are expected. First, we created an original version of the questionnaire taking into consideration the content validity. Thereafter, a web-based survey was conducted among registered participants of a survey company in Japan. The final sample for analysis consisted of 422 participants (176 males and 246 females). The results showed factorial validity through exploratory and confirmatory factor analyses, along with internal consistency and an undistorted distribution of scores for the normal distribution. Thus, the “Self-Esteem Questionnaire in the Teaching Profession (SEQ-TP)” that includes twelve items with the score range of 12 to 84 was completed for future study. Finally, the limitations of this study are discussed, along with possibilities for future research.