To investigate the gender-specific relationship between total bilirubin (TBIL) and fundus arteriosclerosis in the general population, and to explore whether there is a dose–response relationship between them. In a retrospective cohort study, 27,477 participants were enrolled from 2006 to 2019. The TBIL was divided into four groups according to the quartile. The Cox proportional hazards model was used to estimate the HRs with 95% CIs of different TBIL level and fundus arteriosclerosis in men and women. The dose–response relationship between TBIL and fundus arteriosclerosis was estimated using restricted cubic splines method. In males, after adjusting for potential confounders, the Q2 to Q4 level of TBIL were significantly associated with the risk of fundus arteriosclerosis. The HRs with 95% CIs were 1.217 (1.095–1.354), 1.255 (1.128–1.396) and 1.396 (1.254–1.555), respectively. For females, TBIL level was not associated with the incidence of fundus arteriosclerosis. In addition, a linear relationship between TBIL and fundus arteriosclerosis in both genders (P< 0.0001 andP= 0.0047, respectively). In conclusion, the incidence of fundus arteriosclerosis is positively correlated with serum TBIL level in males, but not in females. In addition, there was a linear dose–response relationship between TBIL and incidence of fundus arteriosclerosis.