The enzyme-based degradation of lignocellulose for bioenergy production is an eco-friendly and sustainable approach. This study aimed to elucidate the enzymatic characteristics of endoglucanase (EGL), β-glucosidase (BGL), and xylanase (XYN) from Trichoderma guizhouence NJAU4742, and to explore the potential mechanisms underlying their synergistic degradation of different natural substrates. The results demonstrated that the three enzymes possessed remarkable high-temperature catalytic activity, broad pH adaptability, and responsiveness to different metal ions. The functional group absorption peaks of different substrates were shifted and altered after the synergistic action, particularly for C=O and O-H. Simultaneously, the crystallinity index of wheat straw, soybean straw, rice straw, and corn straw decreased by 7.40%, 2.37%, 20.60%, and 7.67%, respectively, compared to CK (natural straw). Additionally, the dense structure of different substrates was destroyed, and the inner parenchyma began to be exposed after the synergistic action, as observed by SEM. These findings offer valuable theoretical guidance for the development of lignocellulase applications.