Laccase is a blue copper containing enzyme which is capable of catalyzing the oxidation of phenolic compounds to phenoxyl radicals. The purpose of recent study was to find optimum conditions for maximum laccase enzyme production by Pleurotus nebrodensis WC 850 by applying an important statistical technique response surface methodology (RSM) through solid state fermentation of lignocellulosic biomass.  Firstly, screening experiments were conducted in order to select the best substrate for maximum laccase enzyme production among six different lignocellulosic substrates. Wheat straw was selected as best substrate with enzyme activity 145.10 U/mL after 6th day of fermentation. Different physical parameters including pH, temperature, inoculum size, incubation time and moisture were then optimized by response surface methodology under central composite design (CCD). RSM is an effective strategy which explores the optimum operating conditions for multivariable system as well as it also examines the simultaneous, systematic and effective variation of crucial components and determines the possible interaction among higher order effects which results in enhanced enzyme yield. The increase in laccase enzyme activity (171.63 U/mL) was observed at pH 5, temperature 30°C, inoculum size 4mL, incubation time 144mL and moisture 50% after optimization of different factors at different levels. This study reveal that optimization of different factors have significant effect on laccase enzyme production. Moreover, optimization of different parameters through RSM is time saving and good technique in biotechnology.