Enzyme activity was increased during purification which could exert beneficial effect on plants. Methods and Findings: Enzyme purification included extraction, (NH4)2SO4 precipitation, dialysis followed by sequential chromatographies with Sephadex G-75 and Sephadex DEAE A-25. The purified enzyme was characterized with time, pH stability, metal ions, thermostability, and substrate kinetics was determined using guaiacol as substrate. The purification fold for purified broccoli stem peroxidase was 72.83 with 1.5% yield. The optimum time for enzyme activity was 6 min. and it remained stable between pH 4 to 8 having optimum pH of 6 using guaiacol as substrate. The enzyme showed maximum activity at 30°C and remained stable upto 50°C. The Km value of Broccoli Peroxidase was 0.35 m.mol/ml and 33 U/ml for guaiacol substrate by using Lineweaver-Burk graph and similarly using Michaelis Menten graph values was 0.34 m.mol/ml. Metals such as Na+, Ca2+, K+, Mg2+ and Zn2+ exhibited no effect on enzyme activity.