L-Glutaminase, an amidohydrolase enzyme has been a choice of interest in the treatment of leukaemia since the discovery of its anti-tumour properties. Due to its potential use in food industry, clinical applications and analytical applications, many researchers have focused their attention on the production of L-glutaminase. L-Glutaminase is majorly produced by microorganisms including bacteria, yeast and fungi. In the present study, production of L-Glutaminase from fungi, Aspergillus wentii with mixed substrate was reported. Solid state fermentation was followed in the study. Oil cakes which were used as substrate were screened and mixed in different compositions according to simplex lattice mixture design using Design expert v.9.0.5.1. The process parameters and the yield were optimized using response surface methodology.