This work aimed to obtain an ideal mutant strain with higher lipase yield using hydrazine hydrate (HZH) as a novel additive to treat Flavobacterium sp. strain YY25 by supercritical CO2. The survival rate and the positive mutation rate of the tested strain were strongly dependent on the dose of HZH. The treatment by 0.5% HZH in supercritical CO2 (8 MPa, 35°C) for 30 min provided 58.3% of positive mutation rate and an expected mutant strain with about 76.7% increase in lipase yield compared with the wild strain. Possible mutagenesis mechanisms were further explored. The analysis on pH drop of the treated seed liquid was performed to better understand the interaction. Results proved that the induced mutation with enhanced yield of lipase could be achieved by co-mutagenesis of supercritical CO2 and HZH.