Background: Preadolescents, who are in a transitional phase of development, may experience higher exposure to heterosexual interactions while facing higher risks regarding misinformation in sexual knowledge and unsafe engagement in sexual activities. There is a deficiency in the availability of qualified educators and age-appropriate teaching materials for sexuality education in China. Methods: We implemented an animation-based comprehensive sexuality education package among preadolescents aged 9 to 12 years from eight schools in Anhui, China. The first round of intervention included 1,835 participants, lasting 2 months from September to November 2020. A total of 374 participants, accounting for 52% of the intervention group, received a second round of intervention in September 2021. Participants completed immediate follow-up assessment and 1-year follow-up assessment to assess changes in their sexual knowledge, attitudes, and other outcomes. Propensity score matching and difference-in-difference analysis were performed to determine the short- and long-term impacts. Results: Significant improvements were observed for both sexual knowledge and sexual attitudes in the immediate follow-up. There was no significant effect on pornography-seeking behavior or awareness of experiencing sexual abuse. After 1 year, the effect was sustained for sexual knowledge, but slightly declined for sexual attitudes. The second intervention significantly improved sexual knowledge; however, no significant change in sexual attitudes, pornography-seeking behavior, or awareness of experiencing sexual abuse was observed. Conclusions: Our comprehensive sexuality education package was effective in improving sexual knowledge both immediately and 1 year after the intervention. Repeated intervention can be an effective strategy for promoting preadolescent health development regarding comprehensive sexuality education.