The invention belongs to the technical field of compound preparation. The invention relates to a preparation method of phosphatidylglycerol monosodium salt, in particular to a preparation method of phosphatidylglycerol monosodium salt containing two different side chains. The preparation method comprises the following steps: taking D-mannitol as an initial raw material, heating D-mannitol in acetone to generate 1,1,5,6-isopropylidene-D-mannitol; carrying out sodium periodate oxidation and sodium borohydride reduction to obtain iso-propylidene glycerol; carrying out condensation reaction with fatty acid containing an R1 element, then removing acetal protective groups in the presence of acid; selectively protecting primary hydroxyl by using a silane protective agent, reacting secondary hydroxyl with another fatty acid containing a R2 element, removing silane protection, reacting the de-protected product with phosphorus oxychloride and iso-propylidene glycerol, removing acetal protectivegroups by using an acid, and finally carrying out a sodium salt treatment to obtain a final product. The synthesis of phosphatidyl monosodium salt containing two different side chains can be realized,and the production process is simple, environment-friendly and easy to operate.