Glutaminase free L-asparaginase is a vital enzyme because of its anti cancer potential. A potent bacterium isolated from marine environment, producing glutaminase free L-asparaginase using M-9 medium with L-10 asparagine was identified as Pseudomonas plecoglossicida RS1 by 16S rRNA gene sequencing. Statistical modeling was employed to optimize the medium using sugar cane industry effluent as a sole substrate for L-asparaginase production. The enzyme activity of L-asparaginase was increased with M-9 medium containing 0.8% effluent (3.25 ± 0.12 IU/mL) than M-9 medium containing 0.3% L-asparagine (0.73 ± 0.08IU/mL). The apparent K m and V max of the purified L-asparaginase was 2.25 ± 0.61 mM and 15 8.9 ± 0.81 IU/mL/min respectively and the optimal activity of L-asparaginase was at pH 8.5 and 55ºC.