Fatty acyl-CoA Δ9 desaturase (FAD9) is a membrane-bound enzyme anchored in the endoplasmic reticulumthat plays an important role regulating cell membrane fluidity and fatty acid metabolism. The FAD9 sequencehas two transmembrane domains and three amino acid motifs. Purifying the FAD9 protein can be difficultbecause of its membrane structural characteristics. Fish species frequently express FAD9 heterologously. TheChinese mitten crab, Eriocheir sinensis, is an important aquatic species. We designed primers based on the FAD9cDNA sequence from E. sinensis (Accession Number: JQ693685) to obtain the opening reading frame (ORF). TheORF was subcloned into the pCold-TF DNA prokaryotic expression vector to generate the pColdTF-fad9 recombinantexpression vector, which was transformed into E. coli BL21(DE3)pLysS, and FAD9 was expressed successfullyin E. coli BL21(DE3)pLysS using IPTG induction. The sodium dodecyl sulfate-polyacrylamide gel electrophoresisresults showed that the recombinant protein had an approximate molecular weight of 95.10 kD, whichwas consistent with the theoretical molecular weight, and that it was mainly detected in the supernatant. We testeddifferent IPTG concentrations (0.1 mmol/L, 0.3 mmol/L, 0.5 mmol/L, 0.8 mmol/L, 1.0 mmol/L, and 1.2 mmol/L),induction temperatures (35℃, 25℃, 18℃, and 15℃), and induction times (2 h, 5 h, 10 h, 15 h, and 20 h). Theresults showed that when the IPTG concentration was 0.1 mmol/L, expression of the recombinant protein was significantlylower than that at the other IPTG concentrations. Temperatures of 15–18℃ and an induction time of 20 hwere optimal. The highest recombinant protein expression was detected when the IPTG concentration was 0.3 mmol/L,with a 20-h incubation at 15℃. The fusion protein was identified by purification and western blotting. The proteincontained a 6×His-tag, so we used His-tag nickel ion affinity chromatography to purify the protein and ananti-6×His-tag antibody for western blotting. The results showed that the pColdTF-fad9 recombinant protein wassuccessfully expressed in E. coli, and western blotting revealed that the pColdTF-fad9 recombinant protein wasspecifically recognized by the 6×His antibody, indicating that the recombinant protein had antigen activity. Ourstudy provides basic methods to purify and detect the activity of E. sinensis FAD9 and will promote further studyon the functions of FADs.