A lipase gene (AFLB) was cloned from Aspergillus fumigatus GIM3.20. Sequence alignment analysis showed that the AFLB shared 31% amino acid sequences identities with that of Candida antarctica lipase B (CALB) , and both of them were belonged to the CALB-like family. AFLB contained a similar conserved pentapeptide (SWSQG) that the member of CALB-like family shared. Its catalytic triad was supposed to be made up by residues S233,D318, and H361, and the catalytic serine locates in the pentapeptide. What’s more, AFLB hosted a special N-terminal extended sequence, which was a feature of Aspergillus spp. lipase B. AFLB was successfully expressed in Pichia pastoris and purified by affinity chromatography. The result of SDS-PAGE showed that AFLB had partial N-glycosylation, displaying molecular weights of 45-66kDa and 42kDa. Characterization of AFLB lipase showed that the optimum reaction pH was 7.0 and the optimum reaction temperature was 40℃. The optimum substrate for AFLB was p-nitrophenol myristate. AFLB was the homologue of lipase CALB, and might have good industrial application prospect.