The lignocellulolytic enzymes are routinely produced by submerged fermentation using lignocellulosic material, but for more effective production, it would be suitable to precede the production phase on the lignocellulose by propagation phase in the nutrition medium suitable for growth of the fungi. Therefore, the aim of this study was to increase the laccase production by the white-rot fungus Pleurotus ostreatus by two-step cultivation strategy. In the first step, propagation medium was optimized for the maximal biomass growth, the second step included the laccase production by produced fungal biomass in media with the selected lignocellulosic material (pine sawdust, alfalfa steam and corn straw). From our experiments, parameters such as glucose concentration, yeast extract concentration and pH of propagation medium were selected as key factors affecting growth of P. ostreatus. The optimal conditions of propagation medium for maximal fungal growth determined by response surface methodology were: glucose concentration 102.68 g/L, yeast extract concentration 43.65 g/L and pH of propagation medium 7.24. These values were experimentally verified and used statistical model of biomass production prediction was appropriate adjusted. Thus prepared fungal biomass produced in the media with lignocellulose approximately 9-16 times higher concentrations of the laccase in 3 times shorter time than the fungal biomass without propagation phase in optimized propagation medium.