To assess the potential roles of sex steroids in modulating reproductive processes in the Zhikong scallop (Chlamys farreri (Jones and Preston, 1904)), variations in estradiol-17β (E2) and testosterone (T) levels in gonads were examined monthly from January to December 2012 by enzyme-linked immunosorbent assay (ELISA). The mean concentrations of E2 and T in gonads ranged from 75.07 to 666.24 pg/g and from 91.09 to 506.28 pg/g, respectively. Concentrations of E2 were significantly higher in ovaries than in testes, while T concentrations were higher in testes than in ovaries during gametogenesis. Concentrations of E2 in females and T in males increased with development and maturation of gonad, attained the highest value before spawning, and decreased rapidly after spawning. A positive correlation between E2 levels and oocyte diameters (r = 0.743, P < 0.05, n = 25) was observed, suggesting that E2 may play a role in oogenesis. These findings indicate that E2 and T, which are highly correlated with the reproductive cycle, may play an important role in sex determination, sex differentiation, gametogenesis, and spawning in C. farreri.