Abstract: Melatonin is an indole hormone produced by the pineal gland of mammals and humans. It can improve sleep, immune regulation, anti-oxidant and free radical scavenging, anti-tumor, lower blood pressure, regulate heart rate, protection of cardiovascular cells, kidneys, liver and other effects. Melatonin nutrient application in the food industry is greatly limited due to low solubility in water. When melatonin is made into liposomes, it's solubility in polar solvent is increased. However, the current preparing methods of melatonin liposomes are complicated, result in the low entrapment rate, high cost, and use too much organic solvent. From 1970s, supercritical fluids technology has been used in liposomal preparation because of its friendliness, nontoxicity to the environment and its solvent-free liposomes and industrial-scale of liposome production. Phospholipid, cholesterol and melatonin can be solvated by supercritical carbon dioxide concurrently with a certain proportion of ethanol under higher pressure. The liposomes can be obtained when the pressure is reduced to release CO2. The homogeneous super-critical solution is expanded and simultaneously mixed with the aqueous phase to yield liposomes encapsulating the lipid soluble drugs under the lower pressure. The experiment was conducted to enhance the bioavailability of melatonin by improving entrapment rate in this study. The response surface methodology was used to optimize the technical conditions of supercritical carbon dioxide method for the preparation of melatonin liposome. The entrapment rate of melatonin liposome as a major index was optimized based on the ethanol concentration, supercritical temperature and time by response surface methodology using single factor experiments. The applicability of the quadratic-multinomial-mathematical model developed based on the response surface methodology was verified. The results indicated that the highest entrapment rate of melatonin liposome was reached to 83.2% with the supercritical time of 35 min, the supercritical temperature of 50℃, ethanol concentration of 7.5%. Preparing melatonin liposome using supercritical CO2 method was able to be controlled in laboratory. This method is encouraged for promotion in industrial production due to its high entrapment rate, small particle and great stability.