A method for the determination of trans fatty acids C18:1, C18:2 and C18:3 in milk fat was developed by silver ion solid phase extraction-gas chromatography (Ag+-SPE/GC). The fatty acid methyl esters derived from total milk fat were loaded onto an Ag+-SPE cartridge, and then eluted with 9 mL of toluene-hexane (5:95, v/v), 8 mL of toluene-hexane (17:83, v/v), 6 mL of toluene-ethyl acetate (17:83, v/v) and 10 mL of toluene-ethyl acetate (30:70, v/v) in sequence. The fraction from each step was analyzed by GC. The average recoveries of trans fatty acids ranged from 88.4% to 107.2% with the relative standard deviations (RSDs) from 1.2% to 11.9% except trans linolenic acid with the recoveries from 69.9% to 101.0% and the RSDs from 11.0% to 18.1%. Comparing with the traditional methods, the developed approach can avoid the interference of cis and saturated fatty acids. The method was successfully applied to analyze dairy products. Satisfactory results were shown.