Abstract: In this study, a prophenoloxidase (proPO) gene was cloned from hemocytes of red claw crayfish, Cherax quadricarinatus. The open reading frame (ORF) of proPO gene is 1995 bp and encodes a 665 amino acids (aa) protein. The predicted molecular mass of the protein was 75.7 kD with an estimated pI of 6.23. It contained two putative tyrosinase copper-binding motifs with six histidine residues and one thiol-ester-like motif, which showed similar structural features of proPOs from other crustaceans. The amino acid sequence of C. quadricarinatus proPO showed similarity of 68%, 63%, 63% and 59% with that of the proPO of Pacifastacus leniusculus, Homarus gammarus, Homarus americanus and Procambarus clarkii, respectively. The recombinant proPO were expressed in Escherichia coli BL21 with pET-28a expression vector. After protein purification and antibody prepared, the titer of rabbit anti-proPO serum was above 1:12800. The mRNA transcription of proPO and PO activity in C. quadricarinatus were high in hemocytes, hepatopancreas and gills, lower values were seen in other tissues (nerve, heart, intestine and muscle). The immune characterization of proPO gene was studied after Spiroplasma eriocheiris and Aeromonas hydrophila stimulation. After S. eriocheiris or A. hydrophila challenging, the mRNA transcription of proPO gene and PO activity in hemocytes, hepatopancreas and gill of C. quadricarinatus were up-regulated at different times to resist different stimulation. These results indicated that it was potentially involved in the acute response against invading bacteria in C. quadricarinatus.