The fermentation conditions of sophorolipids (SLs) by Candida bombicola O-13-1 with waste fried oil (WFO) as lipid substrates were optimized through orthogonal experiment, and the structures of individual SL components were quantified and identified by HPLC-MS/MS. The results showed that the optimized conditions in shake-flask were determined as follows: agitation 200r/min, carbon source added amount 90g/L, temperature 25℃, inoculum size 12% (volume fraction). The highest yield of SLs of 57.64g/L was obtained at the end of the stationary phase of yeast cell growth, i.e. 204h. The products were composed of 9 kinds of acetylated lactonized and acidic SLs, and the lactonic forms accounted for up to 76% (mass fraction). Moreover, the yield of SLs was as high as 163.28g/L in a 5L automatic fermentor under the optimized conditions of shake-flask with the exception of agitating of 500r/min. The material cost of SLs fermentation with WFO as carbon source reduced by 18% compared with soybean oil, and the resource utilization of WFO could be realized while effectively reducing the production cost of SLs.