Streptomyces ahygroscopicus ZB01 has strong catalytic activity for the regiospecific oxidation of 4aEuro(3)-OH of avermectin to form 4aEuro(3)-oxo-avermectin. A cytochrome P450 gene from S. ahygroscopicus ZB01, cyp107z13, was cloned into pKC1139 to generate pKCZ1 and was transformed into Streptomyces lividans TK54, which does not have the ability to catalyze the conversion of avermectin. CYP107Z13, under the control of an ermE* promoter, was actively expressed in the TK54 recombinant strain as determined by a reduced CO difference spectrum analysis of the crude protein. Analysis of whole-cell biocatalytic activity by high-performance liquid chromatography revealed the recombinant to be able to oxidize avermectin regiospecifically to 4aEuro(3)-oxo-avermectin and CYP107Z13 to be a regioselective oxidase of avermectin. In addition, the whole-cell reaction conditions of the recombinant were optimized. Growth on medium ISP-2 at pH 6 was more conducive for the expression of CYP107Z13 than on medium PYG1 or at pH 7, and active cells of the recombinant strain had higher biocatalytic activity than resting cells.