The invention relates to a method for detecting contents of folic acid and sulfur-containing amino acid in plasma and belongs to the technical field of analysis and detection. The method comprises the following steps: firstly adding a reducing agent for reducing cysteine and homocysteine in plasma; secondly, adding a precipitant for performing albumen precipitation; thirdly, taking supernatant, and adding a reaction solvent and a derivatization reagent for carrying out amino acid derivatization reaction; and finally, adopting a chromatographic column for performing separation and mass spectrum detection determination. According to the method provided by the invention, the folic acid, cysteine, homocysteine and methionine in plasma can be determined at one time, and the interference of other matters is eliminated. The method has the advantages of high precision, accuracy and sensitivity and wide linearity range and can be applied to detection of the contents of the folic acid, cysteine, homocysteine and methionine in large quantity of plasma samples.