PURPOSE: An enzyme with lipase activity, originating from microorganism, is used as a catalyst to effect esterification of a cholesterol with a fatty acid in an organic solvent system at room temperature whereby the title substance is obtained inexpensively through simple process in high yield.

CONSTITUTION: An enzyme with lipase activity, originating from microorganism, such as lipase OF-360 is immobilized on a synthetic resin carrier to provide a catalyst. Then, the catalyst is used to effect the esterification of cholesterol with a fatty acid such as acetic, acrylic or oleic acid in an organic solvent system such as n-hexane or cyclohexane. The reaction temperature is about 20W45°C and the molar ratio of the fatty acid is about 0.5W2 to 1mol of the cholesterol. The objective cholesterol ester is isolated and purified by filtration, crystallization, recrystallization and so on.