In present study, it is aimed to purify peroxidase (EC 18.104.22.168; donor: Hydrogen peroxide oxidoreductase) from leaves of spinach and determine its some biochemical properties. Peroxidase is an oxidoreductase enzyme produced by a number of organisms. Spinach (Spinacia oleracea L.) is an edible flowering plant in the family of Amaranthaceae and is heavily consumed all over the world. Peroxidase enzyme was purified from Leaves of Spinach (Spinacia oleracea L.) by ammonium sulphate precipitation and CM-Sephadex ion-exchange chromatography. K-m (Michaelis constant) and V-max (The maximum velocity of an enzymatic reaction) values were calculated from the Lineweaver-Burk graph for catechol and guaiacol substrates and the substrate specificity of the enzyme was investigated. The results have shown that guaiacol is a better substrate from catechol for this enzyme. Furthermore, for the guaiacol substrate, optimum pH, optimum temperature, optimum ionic strength, stable pH, conditions were determined.