To understand the potential function of pluripotency-associated gene Oct4 in blunt-snout bream (Megalobrama amblycephala), the present study investigated the prokaryotic expression of the recombinant Oct4 protein and generated the rabbit anti-Oct4 polyclonal antibody. Firstly, the expression vector pET32a-MaOct4 was constructed by inserting the C-terminal coding region of Ma-Oct4 into pET32a. Then the expression vector was transformed into Escherichia coli BL21(DE3)pLysS, and the recombinant Oct4 protein was induced by IPTG. After optimization of expression conditions, the protein was largely induced and purified to immunize the New Zealand rabbits. Subsequently, the titer and specificity of the generated antibody were assayed by ELISA and Western blot. The recombinant Oct4 protein was highly induced by 0.5 mmol/L IPTG for 4 h at 37℃. The polyclonal anti-Oct4 antibody effectively recognized the purified recombinant Ma-Oct4 antigen, the induced Ma-Oct4 protein in E.coli, the endogenous Ma-Oct4 protein from the fish embryos, and the overexpressed Ma-Oct4:DsRed fusion protein in HepG2 cells. In conclusion, this research provided an effective antibody to study the potential function of Oct4 in blunt-snout bream.