Sequential solid-state and submerged cultivation with various lignocellulosic biomasses as a substrate for lignocellulolytic enzyme production by Pleurotus ostreatus were assessed by measuring endoglucanase, xylanase, and laccase activities. An unconventional pre-culture method was established by cultivating the P. ostreatus mycelia in a solid substrate medium for an initial fungal growth phase, followed by a transition to submerged fermentation through adding a liquid culture medium. The lignocellulolytic enzymes of P. ostreatus in different fermentation methods revealed wide differences. The higher yields of endoglucanase (3152 ± 139 U/L), xylanase (3064 ± 40 U/L), and laccase (543 ± 21 U/L) were achieved by using the sequential solid-state submerged method compared to conventional solid-state and submerged cultivation. Generally speaking, sequential solid-state and submerged fermentation of cottonseed hull is favorable for laccase secretion, whereas sequential solid-state and submerged fermentation of corncob provides better production of hydrolytic enzymes. These results revealed that the nature of the lignocellulosic biomass and the fermentation method play an important role in the expression of lignocellulolytic enzymes. This indication would be helpful in optimizing the production of integrated industrial lignocellulolytic enzymes.