Abstract: Trichoderma harzianum LTR-2 is a kind of plant disease suppressive fungus. We constructed a expression vector named pC1300-2-G14-C42 of β-1,4-glucanase gene glu14 which was isolated from Bacillus megaterium strain Ap25 and the chitinase gene chi42 was from T. harzianum LTR-2, and used the vector to transform the strain LTR-2 by restriction enzyme mediated integration (REMI). Transformants stability were determined by six successive transfers of conidia on potato dextrose agar (PDA), followed by plating of each isolate onto PDA plates containing 200 μg/mL of hygromycin B. Compared with original strain LTR-2, most transformants changed in growth rate, colonial colour, subiculum density and the conidium quantity. Chromosomal integration was confirmed by PCR amplification and Southern blotting. All transformants not only expressed higher β-1,4-glucanase and chitinase hydrolytic activity, but also high disease suppressive efficacy against tomato late blight (Phytophthora infestans), eggplant damping-off (Pythium ultimum) and cucumber gray mold (Botrytis cinerea) in greenhouse (P< 0.01). Transformant L-15 showed the highest biocontrol efficacy with 91.5%, 94.9% and 83.8%, respectively, against the diseases. Disease suppression rates were increased by 53.5%, 55.9% and 33.5%, respectively in comparison with that of original strain LTR-2. The results indicated that Trichoderma transformants with enhanced biocontrol efficiency could be effectively constructed by inserting β-1,4-glucanase gene glu14 and chitinase gene chi42 into chromosome DNA with REMI method.