Abstract: In this study, the cDNA sequence of Vitellogenin (Vtg) gene in Chinese sturgeon Acipenser sinensis was amplified with the rapid amplification of cDNA end (RACE) method, and its theoretical molecular mass is 196 kD. The antigenic site of the encoded amino acid sequence was cloned, which was used to construct recombinant plasmid and express recombinant protein. The polyclonal antiserum was obtained by immunizing rabbits with purified recombinant protein, and its specificity to Vtg was examined by western blotting. A competitive Enzyme-linked immunosorbent assay (ELISA) was established to detect Vtg in the serum of A. sinensis using antiserum of Vtg as antibody and the purified recombinant protein as antigen. The linear regression of the standard curve was y=–0.2916x+0.6794 (R²=0.9976). The sensitivity of this method was 4.12 μg/mL, and the lowest threshold for detection was 0.3 μg/mL. The inter- and intra-assay coefficients of variation was 2.52% (n=3) and 3.42% (n=3), respectively. The test results of blood sample for the female A. sinensis at different developmental stages showed that the method could be used to monitor the gonadal development of female Chinese sturgeon.