Femtosecond laser irradiation technology was employed for the first time to improve the ascomycin (FK520) yield of Streptomyces hygroscopicus var. ascomyceticus NT2-11, which is an N-methyl-N-nitro-N-nitrosoguanidine (NTG)-induced strain derived from S. hygroscopicus (ATCC14891). The mutant FS35 with high and stable FK520 production capacity was then obtained in the optimal irradiation conditions (25 mW for 6 min) by the Titanium sapphire laser system (810 nm, 76 MHz, 150 fs). The FK520 production capacity of FS35 was 45% higher than that of the parental strain NT2-11. Moreover, under the optimal fermentation conditions, FK520 fermentation titer of FS35 reached 300 mg/L and the intrinsic kinetics of FS35 and NT2-11 were investigated comparatively in 3 phases. The mathematical models provided a good description of FK520 fermentation process for both strains and valuable information for optimizing operation and pilot-plant enlargement research. The comparative studies on parameters of the models confirmed the advantages in production and the decrease of substrate inhibition through femtosecond laser irradiation. Therefore, femtosecond laser irradiation provides a promising way to enhance the production of FK520 in S. hygroscopicus.