Strain improvement was carried out to obtain higher chitinase and protein by inter-specific protoplast fusion between Trichoderma harzianum and Trichoderma viride. Fusant HF9 and parental strains of Trichoderma were compared for chitinase and protein production. 1% of glucose, sucrose and fungal cell wall (Rhizoctonia solani), were used as carbon source for cultivation of Trichoderma and fungal cell wall was the best to induce chitinase and protein. Usage of 0.5% colloidal chitin for the fungal growth under aerated conditions at pH 6.5 and 28°C led to higher chitinase and protein production. In these conditions fusant Trichoderma HF9 in comparison with parent strains had 3-, 2.5- and 1.5-fold increase of total chitinase, specific chitinase and protein, respectively. SDS-PAGE analysis revealed that it had 9 major protein bands with up-regulation compared to parent strains. Amino acid analysis showed that protein of culture filtrate of T. harzianum, T. viride and fusant Trichoderma HF9 had 8, 6 and 10 amino acids, respectively. The results obtained suggested that fusant HF9 could be an integration of T. harzianum and T. viride through protoplast fusion.