The present study aim optimization Pseudomonas extremaustralis production of L-methioninase. The bacterial isolate ability for L-methioninase production was tested on a modified mineral salt M9 agar medium, using phenol red as the pH indicator. Result of optimization L-methioninase production, the cultural and nutrition parameters revealed to the maximum amount of L-methioninase produced by P. extremaustralis were 0.192U\min\ml, obtained after 48h at 35°C of incubation in shaking incubator (150rpm) in culture supplemented with L-methionine mixed with L-glutamine (0.15%), in the presence of glycerol (0.3%) as carbon source at pH6