[Objective] Sophorolipids are biosurfactants with properties such as solubilization, emulsifying, wetting, foaming, dispersing, and reducing surface tension of conventional surfactants, and tolerance to the environment. Starmerella bombicola can produce sophorolipids, but the components of sophorolipids are complex and difficult to separate. In order to obtain the single high-yield acid type sophorolipid, the strain S. bombicola producing only acid sophorolipid was constructed by metabolic engineering.[Methods] The Rec-six gene editing system was developed using the hygromycin resistance gene as a selection marker. Based on this system, the critical lactonase gene SBLE for lactone-type sophorolipid was knocked out to obtain an engineered strain producing only acid-type sophorolipid. The expression cassette of the UGTB gene (glucosyltransferase) was further integrated into the genome of the recombinant strain and the PXA1 gene (peroxisome membrane transporter) was knocked out to construct a metabolic process yeast strain with high acid-producing sophorolipids.[Results] Compared with the original strain, the yield of oleic acid synthase was increased from 20 g/L to 44 g/L, and the lactone type of sophorolipid was undetectable.[Conclusion] The recombinant S. bombicola can effectively increase the yield of acid-type sophorolipids by over-expressing UGTB and knocking out PXA1 and SBLE, for enhanced the production of acid-type sophorolipid by fermentation.