The invention relates to the technical field of lubricating oil, in particular to a preparation method of biological lubricating oil. The preparation method of the biological lubricating oil includes the specific steps that weighed castor oil, epoxy soybean oil, glycerin monostearate, a silane coupling agent, sulfide aliphatic acid ester and thioacetamide are added into a reaction kettle, and are stirred and mixed evenly, then nanometer molybdenum disulfide, zinc dialkyl dithiophosphate and anti-rust composite oil are sequentially added in, uniform stirring is performed, finally, petroleum ether and nano zinc oxide are added in, uniform stirring continues to be performed, and the needed biological lubricating oil is obtained. According to the preparation method of the biological lubricating oil, the castor oil and the modified soybean oil are adopted as base oil, glyceride in the vegetable oil is prone to hydrolysis, the biodegradation rate is high, the viscosity index is high, the viscosity-temperature performance is good, and excellent extreme pressure anti-abrasion performance is achieved because vegetable oil polar molecules form a protective film on the metal surface. The low-temperature fluidity is good, the pour point of the vegetable oil is lower than that of mineral oil, raw materials are easy to obtain, and the cost is low.