Clostridium perfringens is a gram-positive and anaerobic bacterium that is categorized into five types of A-E. Regarding the type of toxins produced, Clostridium perfringens is classified into four types; among them, types B and C strains can produce beta-toxin. Beta-toxin plays a critical role in the development of various diseases in humans and animals and can intoxicate epithelial cells in the intestine. Purification of the beta toxin was performed using affinity chromatography and the obtained product was used for injection into rabbits and production of specific antibodies. Finally, the characteristics of the produced pure toxin were measured. Beta-toxin was purified by one-step affinity purification. Beta-toxin was purified to homogeneity with an overall recovery of 90.1% and specific activity of 4371.5 CU/mg. The purified toxin exhibited a molecular weight of ⁓37KDa, as shown by SDS-PAGE. The Purified beta-toxin was evaluated using standard antibodies by Western blot and ELISA tests. The physicochemical properties of beta-toxin were also determined and optimum pH and temperature values, as well as working buffer for the purification of beta-toxin, were 6, 37^oC, and phosphate buffer, respectively. The K_m and V_max values of beta-toxin were reported to be 9.907 mg/ml and 0.406, respectively. The purified beta-toxin could be applied for different purposes, such as antibody production and determination of the serum rate of anti-toxin antibodies in animals