Comprehensive sexuality education (CSE) aims to foster gender equality in children and young people and has made positive contributions to addressing gender-related challenges. However, little is known about the effectiveness of CSE intervention in weakening gender bias, particularly implicit gender bias. Hence, the present study aimed to explore the potential of a CSE intervention to weaken explicit and implicit gender biases among university students in China. A quasi-experimental intervention design was employed to assess pre- and post-test measurements of explicit and implicit gender biases using a self-report questionnaire and implicit association tests, respectively. Following a 5-month CSE intervention consisting of 36 sessions with university students, an analysis of variance (ANOVA) was performed to compare the differences between the intervention (N = 97) and control (N = 68) groups. The findings revealed that the CSE intervention not only effectively reduced explicit gender bias but also significantly diminished implicit gender bias. The effects of this CSE intervention differed across dimensions and sexes. These results indicate the positive impact of the CSE intervention on gender bias and provide valuable insights for future intervention strategies and evaluation approaches.