Mutagenetic breeding is an efficient technique for the enhancement of lipid productivity from microalgae. In this study, oil-rich microalga Scenedesmus obliquus were treated by Laser-UV composite mutagenesis. Among the 35 mutant strains, X5 was primely screened. Afterwards, a twice UV mutagenizing was operated on X5, and the optimal mutant strain X5-H13 was obtained. The growth rate, dry weight, lipid yield and lipid content of X5-H13 were 0.698×107 cells/mL·d, 0.99 g/L, 0.49 g/L and 48.8% while cultivated in municipal wastewater, respectively, which were increased by 45%, 58%, 109% and 32% than the original strain. The results of the subculture of repeated mutant showed that the biomass and lipid content of strain X5-H13 were up to 0.99 g/L and 48.8%. The growth of each generation was stable. Furthermore, the random amplified polymorphic DNA analysis indicated that the mutant strain X5-H13 was different from the starting strain, with their genetic similarity coefficient value of 0.815.