The biocatalysis of partially purified chlorophyllase from Phaeodactylum tricornutum in an organic solvent medium was investigated. The optimum amounts of water and organic solvent required for the chlorophyllase-catalyzed hydrolytic activity were measured in a wide range of solvents, including acetone, ethanol, propanol, butanol pentanol, hexanol, toluene, pentane, hexane, octane, and heptane as a function of their hydrophobicities. The logarithm of partition coefficient (log P) was used to define the quantity of solvent polarity. The amount of aqueous buffer required for the enzymic activity was lower in non-polar solvents (log P > 1.8) than polar ones (log P < 1.8). The V-max values for the hydrolytic activity of chlorophyllase in water-miscible solvents (log P < 0.8) were higher than those in water-immiscible solvents (log P> 0.8). The hydrolytic activity was decreased by approximately 12 times by increasing the carbon chain of alcohol from two to five. The results also indicated that phytol has a non-competitive inhibitory effect on the chlorophyllase activity in reaction media containing acetone, and activatory effect in that containing hexane.