Globally, fungi and fungi-like organisms, with over 8,000 species, cause more plant diseases than any other group of plant pests. Chitinases possess an anti-fungal role in disease resistance. They represent the pathogen-related protein group that cleaves β-1,4-glycoside bond of chitin present in the cell wall of filamentous fungi by catalysing the hydrolytic cleavage. In this study, we have amplified 672 base pair (bp) coding regions of the plant chitinase gene of Hordeum vulgare, over-expressed in Eschercia coli (E. coli) host BL21 DE3 strain by 0.5mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. In western blotting, the recombinant chitinase protein transferred on nitrocellulose membrane was detected at 26 kDa with histidine-tagged antibody reactions. By applying the nickel charged sepharose column affinity chromatography method, a single purified band of recombinant chitinase protein was found at 26 kDa in SDS-PAGE. The purified recombinant chitinase protein during the agar well diffusion method inhibited the growth of three phytopathogenic fungi in a significant way; Alternaria alternate, Rhizoctonia solani and Fusarium oxysporum in qualitative in-vitro antifungal assays. The purified recombinant chitinase protein showed up to 82.6% reduction in A. alternate hyphal growth; 77% reduction of hyphal growth in Rhizoctonia solani and 78% reduction of Fusarium's hyphal growth in invitro quantitative antifungal assays, thus depicting a strong antifungal potential. Conclusively, plant derived chitinase genes are superior in protecting the crops from phytopathogenic fungi if cloned in a suitable plant vector and expressed.