Purpose: In the pre-school period, an active sense of curiosity, the most important developmental feature of children, is directed toward sexuality. In this context, it was aimed to develop a questionnaire to measure the sexual knowledge of children and to develop a sexuality education program based on the information level on sexual issues, and cognitive and emotional readiness of children aged 60-72 months. Research Methods: In the first stage, a questionnaire aimed at identifying sexuality knowledge level of 45 items was designed, and in the second stage, contents of 11 sessions on sexuality education program were created. For the content validity study of the questionnaire and the sexuality education program, 10 specialists were involved in data analysis. To determine the feasibility and the scoring criteria for the sexual knowledge questionnaire, a pilot scheme was carried out with 18 children aged 60-72 months attending the preschool institution. Findings: After consultation with a specialist opinion, a questionnaire for sexuality knowledge composed of 45 items was reduced to 24 items and the sexuality education program composed of 11 sessions was reduced to 8 sessions. According to the research findings, the content validity results of both the sexuality education program (KGI[greater than or equal to]KGO (0.62)), and the questionnaire on sexuality knowledge level (KGI (0.63)[greater than or equal to]KGO) were statistically significant. Implications for Research and Practice: It is thought that the sexuality education program prepared for pre-school children is suitable for pre-school programs. It will also contribute to the understanding of how the use of the questionnaire for sexuality knowledge can assess the effectiveness of sexuality education programs.