To improve the yield of daptomycin by Streptomyces roseosporus, a method of rational screening of He-Ne Laser and N-methyl-N-nitro-N-nitrosoguanidine (NTG)-induced mutants was employed in this work. Under the optimal mutagenesis conditions (NTG of 0.3 mg/L, 40 min; irradiation at 15 mW, 15 min), two steps of combined mutations were conducted. Screening of mutants was done according to the individual resistance to n-decanoic acid and daptomycin. A mutant strain LC-54-16, with the highest daptomycin production ability of 616 mg/L was obtained, which was over 5 times higher than the wild strain LC-52-6. The transcription levels of the main dpt genes in the mutant were approximately five times higher than those in the wild. The superiority of the mutant to the wild strain was further proved by the comparative studies on the kinetics of the mutant and the wild. The decrease of the inhibition of substrate and product was further confirmed, as well. It was concluded that the method of rational screening of He-Ne Laser and NTG-induced mutants could efficiently improve the daptomycin production ability of S. roseosporus.