Fatty acyl-CoA Δ6-b desaturase is a membrane-bound enzyme, and it is the rate-limiting enzyme in the biosynthetic pathway of highly unsaturated fatty acids (HUFA). Fatty acyl-CoA Δ6 desaturase can catalyze the first step of the desaturation in the HUFA pathway, and it can convert linoleic acid(LA, 18:2n-6) to Gamma linolenic acid(GLA, 18:3n-6), convert α-linolenic acid(ALA, 18:3n-3) to Stearidonic acid(SDA, 18:4n-3), and it also can convert Eicosapentaenoic acid (EPA, 20:5n-3) to Docosahexaenoic acid (DHA, 22:6n-3) with other enzymes. In this study, fatty acyl-CoA Δ6-b desaturase gene was cloned from Eriocheir sinensis using reverse transcriptase polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends (RACE). The full-length cDNA sequence of fatty acyl-CoA Δ6-b desaturase is 2 310 bp, including a 1 326 bp open reading frame (ORF) and codes a protein of about 442 amino acids. The molecular weight is 50.86 ku, and the isoelectric point is 8.47. GenBank accession number of this gene is KP876058. A homology analysis using BLASTn and BLASTx revealed that the fatty acyl-CoA Δ6-b desaturase gene shared 76% identity with fatty acyl-CoA Δ6 desaturase gene of E. sinensis. The ORF of fatty acyl-CoA Δ6-b desaturase gene was subcloned into the prokaryotic expression vector pCold-TF DNA, to generate recombinant expression vector pColdTF-fad6b, which was then transformed into the expression E. coli BL21(DE3)pLysS. Experiments showed that the fatty acyl-CoA Δ6-b desaturase was successfully expressed in E. coli BL21(DE3)pLysS by the IPTG induction and at the temperature of 15, and the concentration of IPTG was 0.3 mmol/L. The result of SDS-PAGE analysis showed that the recombinant protein had an approximately molecular weight of 105.86 ku which was consistent with the theoretical molecular weight, and the target protein was mainly detected in supernatant. As the purpose protein contains a 6×His-tag, we have chosen His-tag nickel ion affinity chromatography column for recombinant protein purification and anti-6×His-tag antibody for Western-blotting experiments, and results showed that pColdTF-fad6b recombinant protein was successfully expressed in E. coli. The Western-blotting revealed that recombinant protein pColdTF-fad6b had specifically been recognized by the 6×His antibody, indicating that the recombinant protein had antigen activity. Our report provides a new fatty acyl-CoA Δ6 desaturase gene, FAD6b. It may offer a basic method for purification and activity detection of E. sinensis FAD6b, and promote further study of the FADs function.