The expression of the recombinant caffeinyl-coenzyme A O-methyltransferase gene (CCoAOMT) in E. coli cells was optimized and the enzyme was purified by Ni-NTA affinity chromatography. The enzymatic reaction was carried out in vitro with epigallocatechin gallate (EGCG) as substrate, and EGCG3"Me, the reaction products, were analyzed by HPLC. As a result, CCoAOMT was highly expressed in E. coli BL21 and the best induction conditions of recombinant CCoAOMT was 0.5 mmol/L IPTG at 37 ℃ for 4 h. The recombinant protein was purified by Ni-NTA affinity chromatography. The recombinant CCoAOMT could catalyze EGCG to EGCG3"Me in vitro and the optimal reaction conditions include substrate EGCG of 0.05 mmol/L, pH of 4 and temperature of 37 ℃.