To achieve high value-added utilization, vinasse was chosen as raw material for l-lactic acid production. Alkali methods, microwave were used to treat vinasse and the optimum parameters during the process were investigated. The results showed that the best pretreatment way was the combination of alkali concentration of 8% and microwave power 700W, corresponding maximum lactic acid concentration was 17.5g/L, higher than those of the independent method utilized, the fermentation time could be one day earlier than other methods. The conversion rate of preprocessed vinasse to L-lactic acid and utilization rate of cellulose and hemi-cellulose could reach 23.8% and 71%, respectively. The optimized pretreatment could effectively realize hydrolysis of vinasse and make it an ideal sources for further utilization.