Lignocellulosic biomass is widely used in the field of laccase production because it has the advantages of low price and easy availability. Thus, a comparative analysis was performed of the laccase secretion ability of five white-rot fungi in submerged fermentation using single or mixtures of lignocellulosic biomass. Maximum laccase activity of Trametes gibbosa An 360, Vanderbylia fraxinea An 369, Perenniporia pyricola Han 202, Coriolopsis trogii Han 474, and Trametes versicolor Han 1504 was 55.83 ± 0.28 U/L on the mixture of corncob and cottonseed hull, 77.96 ± 1.60 U/L on corncob, 443.33 ± 15.49 U/L on corncob, 686.57 ± 16.49 U/L on corncob, and 162.04 ± 11.33 U/L on cottonseed hull. The mixed lignocellulosic material effectively improved the laccase activity of T. gibbosa An 360 compared with other fungal strains. However, the presence of corncob contributed to the secretion of laccase activity for V. fraxinea An 369, P. pyricola Han 202, and C. trogii Han 474. Meanwhile, cottonseed hull was conducive to the secretion of laccase of T. versicolor Han 1504. Laccase activity of P. pyricola Han 202 was very stable throughout most of the fermentation time. The results from this study provide new methods and increase fungal strains to improve laccase activity.