This work is concerned with the purification and characterization of the cholesterol oxidase enzyme synthesized by probiotic bacteria insulated from raw cow's milk. The bacterial isolate was genotyped as Lactiplantibacillus plantarum by 16S rRNA and deposited with GenBank under accession number MW242720. The L. plantarum MF1 cholesterol oxidase enzyme was purified using acetone and Sephadex G 100 column chromatography, yielding enzyme recovery and purification folds of 19.3% and 4.2, respectively. The purified cholesterol oxidase appeared as a single protein band at 51 kDa using the SDS-PAGE technique. For the maximum activity of the purified CHO enzyme, the optimal conditions were a 30-minute' incubation period at 30°C, a substrate concentration of 150 μL in the reaction mixture, and an enzyme concentration of 100 μL in the reaction mixture. The purified cholesterol oxidase was activated by metal ions (CuSO4), whereas it was inhibited by CaCl₂, MgSO₄, ZnSO₄, and MnCl₂. After storage at -20°C for 4 days, the purified enzyme retained 57% of its initial enzyme activity. The purified enzyme demonstrated an active effect on serum human cholesterol samples, resulting in a 29% reduction.