Doublesex (Dsx) and its homologous genes are important regulators of sex differentiation in the animal kingdom, which determine the differentiation of animal somatic and germ cells. To elucidate the function of Doublesex(PcDsx)gene in Procambarus clarkii, the cDNA sequence was cloned by the rapid amplification of cDNA ends (RACE) and the expression of PcDsx was determined by quantitative real-time PCR (qRT-PCR). The full-length PcDsx cDNA is 1584 bp, with a 243 bp 5′ untranslated region, a 765 bp open reading frame (ORF) (coded 254 aa) and a 576 bp 3′ untranslated region. The deduced protein of PcDsx cDNA was 254 aa. The predicted PcDsx protein was found to contain a conserved DM domain. The phylogenetic analysis revealed that the PcDsx DM domain has a high similarity to the SvDsx DM domain of Sagmariasus verreauxi. The results of gene expression analysis showed that PcDsx was widely expressed in various tissues of the adult crayfish. The relative expression of PcDsx was the highest in antennary glands, followed by muscle and gonads. It was found that the expression of PcDsx in various tissues of adult female were significantly different from that of male corresponding tissues. Within the early development of P. clarkii, the expression level of PcDsx reached a peak at the third day after day after hatching, while the highest expression of PcDsx in male and female juveniles appeared at 41 and 115 day after hatching, respectively. Besides, the expression of PcDsx in male and female juveniles also showed significant differences.