In this work, a mutant MX3004 with improved micronomicin (MCR) production was derived from Micromonospora sagamiensis ATCC21826, which was treated with femtosecond laser under the optimized irradiation conditions of 75 mW and 180 s, with a maximum of positive mutation rate of 17.8 % and the mortality rate of 69.2 %. A novel high-throughput method was established using microplate reader by quantifying the concentration of MCR for efficient screening of positive mutant from large numbers of mutants. Consequently, MX3004 displayed the highest MCR production capacity of 126 U/ml and a stable heredity (ten generations). Moreover, under the optimal fermentation conditions in a 7.5 l fermenter, the MCR production of MX3004 reached the maximum of 263 U/ml, which was increased by 484 % compared with the parent strain. The results suggest that femtosecond laser is a suitable method for the MCR production improvement and the screening method has a great potential application for aminoglycoside antibiotic production.