The 3α-hydroxysteroid dehydrogenase gene (3α-hsd)from Comamonas testosteroni was amplified by PCR from the genomic DNA of Comamonas testosteroni. It was cut out from PCR product by the digestion of the restriction enzymes NdeⅠ/BamHⅠ and was cloned to plasmid pET-15b again. The recombinant plasmids pET-15b with proper orientation were identified by the analysis of restriction enzymes and DNA sequencing. Then the host bacteria containing recombinant plasmids pET-15b with proper orientation grew with IPTG induction. The total cell lysate of the host bacteria was extracted for SDS-PAGE and the determination of its enzymatic activity. Afterwards the recombinant protein with an N-terminal His tag sequence was purified by affinity chromatography on a Ni²⁺-Sepharose column. The recombinant plasmid pET-15b with proper orientation containing the 3α-hsd gene was selected and the fusion protein was overexpressed in the host bacteria of E.coli BL21(DE3) pLysS. The enzymatic activity of the crude extracts was up to 2.45×10⁵ U/L.The fusion protein could be purified in one step using metal chelate chromatography to homogeneity as judged by SDS-PAGE. And the recovery rate was about 68%. The above work has laid a necessary foundation for the construction of a enzymatic cycling method to determine the serum total bile acids.