The invention relates to a preparation method and application of trans-folic acid. The preparation method comprises the following steps: using p-aminobenzoyl glutamic acid, triaminopyrimidine sulfate and trichloroacetone as raw materials for chemical synthesis to significantly increase the content of a coarse product of trans-folic acid; then, separating by a preparative chromatography to obtain the trans-folic acid with the purity greater than 95%. The research of the invention is beneficial to further research on a production mechanism and a control method of the trans-folic acid, and can effectively improve the control level of the quality of folic acid.