To investigate the sexual preference of zebrafish (Danio rerio) during the reproductive period and the role of 17β-estradiol (E2) in sexually dimorphic preference, sexual preference tests were conducted on male and female zebrafish during the reproductive period. Subsequently, female zebrafish were treated with letrozole, and male zebrafish were treated with E2. After 28 days, their sexual preferences during the reproductive period were detected, along with endogenous E2 levels and brain serotonin (5-HT)/dopamine (DA) concentrations. The results show that both male and female zebrafish preferred the opposite sex during the reproductive period before drug treatment, but lost this sexual preference after treatment. Following treatment, endogenous E2 levels in females were significantly decreased, accompanied by a significant increase in brain 5-HT levels. In males, endogenous E2 levels were significantly elevated, while brain 5-HT and DA levels were significantly reduced. These findings indicate that sexual preference during the reproductive period in zebrafish exhibits sexual dimorphism, which may be closely associated with endogenous E2 levels and brain 5-HT/DA concentrations.