The invention discloses a ruminant trans-fatty acid preparation method, which comprises: (1) carrying out a saponification reaction on butter and a sodium hydroxide ethanol solution under a 85-90 DEG C water bath condition, adding a sulfuric acid solution, stirring under a 45-50 DEG C water bath condition for 20-30 min, dissolving, standing, and taking the upper layer oily matter to obtain butter free fatty acid; (2) adding urea into absolute ethyl alcohol according to a certain proportion, carrying out rotary reflux at 70-85 DEG C, then adding a certain amount of butter free fatty acid, carrying out reflux at 77 DEG C, and carrying out inclusion at-5 DEG C; performing vacuum suction filtration to obtain filtrate, performing rotary evaporation on the filtrate at 65 DEG C, removing ethanol, dissolving with deionized water at 60 DEG C, extracting upper oily matter, drying with anhydrous sodium sulfate, and filtering to obtain the ruminant trans-fatty acid product. The preparation method of the ruminant trans-fatty acid is simple in process, high in operability, low in production cost and energy consumption and easy to realize large-scale continuous production.