The hepatopancreas of Procambarus clarkii provides lipids and other nutrients for ovarian development and contains hormones such as estradiol related to ovarian development. During the development of the P. clarkii ovary, the hepatopancreas’ tissue structure and fatty acid content undergo corresponding changes. A controlled indoor experiment was conducted to investigate the effects of exogenous estradiol injection on the tissue structure and fatty acid content in the hepatopancreas of P. clarkii. The experiment consists of three experimental groups and one control group. The P. clarkii in the experimental group were injected with 5, 0.5, and 0.05 μg/(g body weight) of estradiol once every 5 days for 5 times and with normal saline injection as the control. After 25 days, the content of fatty acids and changes in tissue structure in the hepatopancreas were measured. The results showed that the main fatty acids in P. clarkii hepatopancreas were C18:4n, C20:3n, and EPA, and polyunsaturated fatty acid (PUFA) content was 57.17%±4.76%. Injecting estradiol mainly affected the content of C16:0, C16:1n, C18:0, C18:1n9, C18:2n, C18:3n6, C18:3n6, C18:4n, C20:1n, C20:2n, C20:3n, C20:4n3, C20:5n3, C22:n (n=1-4), C22:5n, and C22:6n in the hepatopancreas. Injecting estradiol significantly reduced the total content of PUFA in hepatopancreas with the reduction of C18:3n6, C18:4n, C20:3n, C20:4n3, C20:5n3 C22: n (n=1-4) (P<0.05); Estradiol injection significantly increased the total content of monounsaturated fatty acid (MUFA) in hepatopancreas with the increase of C18:1n9, and C20:1n (P<0.05); Injection of estradiol (0.05 μg/g) significantly increased the total content of saturated fatty acids (SFA) in the hepatopancreas (P<0.05) with the increase of C16:0 and C18:0 (P<0.05). Injecting estradiol can increase the volume and quantity of hepatopancreatic B cells, and the 5 μg/g group had the most obvious changes.