The production, characterization and partial purification of hydrocarbon degrading peroxidase from di-culture of Rhizopus and Saccharomyces species was assessed. The fungi were isolated from hydrocarbon polluted soil and identified using standard microbiological and biochemical techniques. Fermentation was carried out for enzyme production and the extracellular extract was precipitated with 50% ammonium sulphate salts and further purified by gel filtration\using sephadex G100 gel and by dialysis. The enzyme activity and stability was assayed against varied pH and temperature. Four genera of fungi which included Saccharomyces, Penicillium, Rhizopus and Aspergillus species were isolated from the crude oil polluted soil of which Saccharomyces sp. and Rhizopus sp. produced peroxidase. Maximum enzyme production occurred on day 8. The enzyme activity was optimum at a pH of 4.5 and temperature of 50°C. The Km and VMAX were 1.8 mg/mL and 20.3 μmol/min respectively, against the 5 mM of O-dianisidine substrate. Stability studies showed that the peroxidase was stable in the presence of Ca 2+ , Mg 2+ and Co 2+ , with Ca 2+ showing the best stability. In conclusion, peroxidase was partially purified from di-culture of Saccharomyces and Rhizopus species isolated from crude oil polluted soil. The presence of this enzyme in crude oil polluted soil may suggest its implication in the degradation of crude oil. Further studies need to be done to assess the potential of this enzyme in the degradation of crude oil.