Abstract: In order to quickly and efficiently screen out Aspergillus oryzae strains of high-yielding Rhizomucor miehei lipase (RML), the spores of Aspergillus oryzae (A. oryzae) were mutated by atmospheric room temperature plasma (ARTP) mutagenesis technology. The dual indicator method for high-throughput detection of RML enzyme activity was optimized, and a 24-well plate high-throughput culture system of A. oryzae was established.Through high-throughput screening of 1,200 wells (36,000-48,000 spores) after mutagenesis, 4 high-yielding mutants, which was designated Ⅰ 7-D6-7, Ⅱ 4-4B-5, Ⅲ 3-5C-1 and Ⅴ 7-6C-3, were finally obtained. In shake flask cultures, the enzyme activities of mutants reached 265.98, 240.90, 253.52 U/mL and 293.50 U/mL, which was increased by 50.68%, 36.47%, 43.61% and 66.27%, respectively, compared to the original strain (176.52 U/mL) .