The yeast Starmerella bombicola CGMCC 1576 can produce abundant sophorolipids (SLs) including almost equal proportion of acidic and lactonic SLs. In this study, a monooxygenase MoA responsible for the metabolism of a sophorolipid molecule, C18: 2 diacetylated acidic sophorolipid (C18: 2 DASL), was identified, through genomic analysis, protein modeling, and gene knocking out strategy. The yield and compositions of SLs produced by the deletion mutant Delta moA changed dramatically. In HPLC chromatogram, the UV absorption area of C18: 2 DASL (one major acidic sophorolipid component) increased from 9.84x10(6) mAUxs to 34.26x10(6) mAUxs by an increase of 248.17 % when oleic acid was used as hydrophobic carbon source. Moreover, when linoleic acid was used as hydrophobic carbon source, the content of C18: 2 DASL component produced by the overexpressed strain Peno:: moA decreased significantly compared with that of wild type and Delta moA. Furthermore, the MoA enzyme was heterologously expressed in Escherichia coli JM109 (DE3) with a recombinant plasmid named pMAL-c2x- moA, and the purified enzyme was obtained through a maltose-binding protein (MBP) affinity chromatography column. The purified C18: 2 DASL and C18: 1 DASL were applied to be catalyzed by MoA enzyme, respectively; it turned to be that C18: 1 DASL still remained in the MoA reaction system, but C18: 2 DASL disappeared.