Box- Behnken experimental design for cultural optimization engages a specific study on the area of individual factors such as temperature, pH, Agitation, Inoculum size and Incubation time. Production of laccase increased by modification of cultural conditions. In this study, RSM-BB was used for optimizing culture media for maximizing laccase production by using Pleurotus ostreatus in batch fermentation. The optimized cultural conditions increased two fold of laccase production. 46 experiments runs were attained for optimum enzyme production and the coefficient of determination was 99.1% with adjusted R2 value of 98.3 and predicted with 96.5 for enzyme production from Pleurotus ostreatus PKN04. For the biomass production the R2 was 98.6%, adjusted and predicted R2 were 97.5% and 94.65% respectively. This shows the significance of the model and effective production of enzyme from Pleurotus ostreatus