Wnt16 is a member of the nonclassical wnt signaling pathway. Recently, a large number of studies have shown that wnt16 is related to the development of hematopoiesis and skeleton in zebrafish. To elucidate the mechanism of wnt16 in regulation of hematopoiesis, the whole ORF (open reading frame) sequence of wnt16 gene in zebrafish was amplified by RTPCR (reverse transcription PCR), and the eukaryotic expression vector pCAGGSP7/wnt16 was constructed. Then BALB/c female mice immunized with the expression vector to generate antiWNT16 specific polyclonal antibody in zebrafish. The results showed that the antiWNT16 antiserum prepared by immunizing DNA could specifically recognize WNT16 protein. The successful preparation of zebrafish WNT16 antibody lays an important foundation for further study.