To investigate the function of the pluripotency transcription factor Nanog in fish, this study examined the prokaryotic expression of the recombinant Nanog protein and generation of the rabbit anti-Nanog polyclonal antibody in blunt-snout bream. Firstly, MaNanog coding sequence was obtained from the ovary organ, and the full-length open reading frame or the partial fragment containing the homeodomain was inserted into the pET32a vector, respectively. Then the vectors were transformed into Escherichia coli BL21(DE3)pLysS, and the recombinant Nanog proteins were induced by IPTG. After optimization of expression conditions, the MaNanog-S protein was induced on a large scale and applied to antibody generation. Subsequently, the specificity of the antibody was examined by ELISA and Western blot. Results showed that the recombinant Nanog protein was highly expressed with 0.5 mmol/L IPTG induction for 4 h at 37 ℃. The polyclonal antibody could identify effectively the induced Nanog protein in E. coli, the endogenous MaNanog protein in adult organs, and the ectopic expressed MaNanog proteins in HepG2. Taken together, these results provided the research methods for prokaryotic expression of protein and preparation of specific antibodies, and also provided an effective tool to investigate the function of Nanog gene in fish.