Among Chinese soft-shelled turtles, Pelodiscus sinensis, males have a richer nutritional value and higher market price than females. All-male offspring were obtained by 17β-estradiol (E2). However, the molecular mechanisms of E2 inducing sexual reversal remain unclear. In this study, we cloned estrogen nuclear receptors (ERs) from P. sinensis and investigated their expression profiles. We examined the responses of ERα and ERβ after treatment with different concentrations of 1.0, 5.0, and 10 mg/mL E2. ERs showed abundant expressions in the adult gonad, ERα for ovary, and ERβ for testis. E2 can up-regulate the expression of ERα, which showed a remarkable increase while the promotion of ERβ was unobvious. They reached a high level at stage 17 after the treatment of E2, genes of the female-related genes Rspo1, Wnt4, β-catenin, Foxl2, Cyp19a1, and Sox3 exhibited a significant raise at stage 17 with the increase in the concentration of E2 while the male-related genes Sox9, Dmrt1, and Amh were significantly inhibited. Our study cloned the full length of ERs and analyzed their structures and expressions, laying a foundation for the further study of the effect of estrogen on sex determination.