In the current study, thermo-tolerant fungal isolates were screened for their ability to metabolize L-methionine by L-methionine γ-lyase (MGL). Among 63 fungal isolates, fifteen isolates exhibited ability to MGL induction as evidenced by qualitative assay method using phenol red. Then, quantitative assay method indicates that fungal isolates coded as 26 & 37 were the most potent for MGL production. Fungal mycelium treatment under the sonication process proved that isolate No. 37 could be release MGL with maximum activity, meanwhile isolate No. 26 has been produced MGL extracellularly under the same culture conditions. Thereafter, characterization and identification of these two isolates was carried out by morphological, microscopic examination and molecular techniques. In consequence of this characterization, isolate No. 26 & 37 were identified as Aspergillus fumigates and Rhizomucor miehei respectively. Different culture conditions were screened by Plakket-Burmn design to define the significant parameters that affect the induction of MGL of the two fungal strains. Using the response surface method model, independent culture parameters were optimized through a central composite design (CCD) to maximize the induction of extracellular and intracellular MGL by A. fumigatus and R. miehei. Statistical optimization using Response surface methodology (RSM) revealed that yeast extract, incubation period and temperature were significant factors for MGL production by R. miehei where MGL was 8.27 U/mg. Additionally, glycerol, PH, period and inoculum size were the most efficient factors affect MGL by A. fumigatus where MGL was 12.37 U/mg.