The effects of polarity of various organic solvents, including acetone, ethanol, and propanol, used in a biphasic organic system, on the hydrolytic activity of a partially purified chlorophyllase from Phaeodactylum tricornutum were investigated. The different concentrations of each polar organic solvent, from 0 to 40%, were added to a mixture (45:55, v/v) of hexane and a buffer solution of Tris–HCl (20 mm, pH 7.5). The most appropriate concentrations of acetone, ethanol, and propanol for the hydrolytic activity of chlorophyllase were 12.5, 5.0, and 2.5%, respectively. The results indicated that the optimum reaction time for the chlorophyllase activity in the biphasic system decreased from 7.0 h to 3.0, 5.0, and 5.0 h, respectively, upon the addition of an appropriate amount of acetone, ethanol, or propanol. The Vmax and Km as well as the inhibitory effect of phytol on the chlorophyllase activity in the biphasic organic system containing a polar organic solvent were also investigated.