A robust process of purification, characterization, and application of endoglucanase from the agro-industrial waste was performed using solid state fermentation (SSF). Trichoderma harzianum as a micro-organism and wheat straw as a growth supportive substrate were used in SSF under pre-optimized conditions. The maximum activity of 480 ± 4.22 U/mL of endoglucanase was attained when a fermentation medium was inoculated using 10% inoculum size and 3% substrate concentration with pH = 5.5 at 35 °C for an optimized fermentation period. In comparison with crude extract, enzyme was 1.83-fold purified with a specific activity of 101.05 U/mg using Sephadex-G-100 column chromatography. Sodium dodecyl sulfate (SDS) poly-acrylamide gel electrophoresis revealed that the enzyme exhibited a low molecular weight of 43 kDa. The purified enzyme displayed maximum activity at pH = 6 and a temperature of 50 °C, respectively. The maximum activity (Vmax) of 156 U/mL and KM value of 63 µM were observed. Ethylenediaminetetraacetic acid (EDTA), SDS, and Hg2+ inhibited enzyme activity, while Co2+ and Mn2+ enhanced enzyme activity at 1 mM concentration. The maximum substrate affinity and specific activity of biosynthesized endoglucanase revealed that it can be potentially useful for industrial applications.