Preparation method of levo-5,10-methylenetetrahydrofolate

The invention relates to a preparation method of a levo-5,10-methylenetetrahydrofolate. By taking a p-toluenesulfonic acid as a resolving agent, tosylate is subjected to chiral resolution according to the difference between the solubilities of the tosylate in water by using levo-/dextro-5,10-methylenetetrahydrofolates, so that by using the method disclosed by the invention, a levo-5,10-methylenetetrahydrofolate with a specific rotation of [alpha] 20D>=36 o, (c = 1,0.04 N NaOH) can be obtained, wherein the (6R)-5,10-methylenetetrahydrofolate content is greater than or equal to 89%.