Background: Crustaceans often exhibit significant sexual dimorphism during their growth process. However, their sex determination system is relatively complex, and still lacks of related studies that involved in sex determination and differentiation. Results: In the present study, the gene of Doublesex (Cqdsx) was identified and characterized for the first time in the redclaw crayfish, Cherax quadricarinatus. The full-length cDNA was 1271 bp, comprising a 155 bp 5’-untranslated region (5’-UTR), a 885 bp predicted open reading frame (ORF) encoding 294 amino acid polypeptides, and a 231 bp 3’-UTR. The deduced amino acid sequence of Cqdsx was predicted to contain a highly conserved DM domain and shared nearly 50% identity to DM-peptides from other species. The results of quantitative Real-time PCR in various tissues revealed that Cqdsx was strongly expressed in gonads, while was almost undetectable in gill, heart, hepatopancreas, muscle and intestine. Comparing expression level in different embryonic stages found that Cqdsx was gradually increased with the development of the embryos. In situ hybridization to gonad sections showed that intensive hybridization signals were mainly observed in oocytes and ovarian lamellae and weak signals were detected in spermatocyte. Additional, Cqdsx gene exhibited the higher transcript levels in the early stage of ovarian development. Furthermore, RNAi-targeting Cqdsx silencing induced a decrease of Cq-IAG trascripts, which regulated the male sexual differentiation in crustacean. Conclusion: DM-domain genes play an important role in the sex determination and differentiation among animal kingdom. The full-length cDNA of Cqdsx in C. quadricarinatus was isolated and characterized. Our findings strongly suggests an essential role for Cqdsx in the female ovarian development/differentiation of the redclaw crayfish. These data may provide us a better understanding of sex determination in C. quadricarinatus.