Horseradish peroxidase (HRP) is the group of enzymes that catalyze the oxidation of a compound by peroxide. Horseradish peroxidase is the universal choice of reagent in molecular level experiment, diagnostics, sewage treatment and in Biosensor. Horseradish peroxidase is separated through traditional down streaming method, which usually results in low yield and high cost. Liquid-liquid extraction (LLE) using organic/aqueous phase is not the choice of separation in biotechnology mainly due solubility and protein denaturation problems.Aqueous Two Phase System (ATPS) adapts the extraction through phase separation and is considered as single step isolation protocol. TritonX-100 and Tween 20 were used as surfactants, and efficiency were calculated.Aforward extraction yield of 72%and activity 890 U/mg was obtained for Triton X-100/ Toluene at pH 6, salt concentration 0.15M, phase ratio 1:1 and surfactant concentration 15mM. In Tween 20/ toluene system there was only 58% yield in 10µl surfactant concentration. For back extraction amaximumyield of 13%and activity of 648 U/mg obtained in Triton X-100/ Toluene system.ATPSwas found to be superior to traditional methods both in terms of yield and continuous process. Isolated peroxidase was checked for its purity using SDS-PAGE, which clearly displays 3 bands correspond to the standard marker and commercial peroxidase.Gas chromatography analysis conforms the presence of peroxidase however 11 more peaks indicating presence of other proteins and enzymes. It is suggested to optimize the techniques for continues separation and concentration of the peroxidase.