The invention relates to a preparation method of DHA (which relates to an unsaturated fatty acid. The invention provides a preparation method of a fully synthetic medium that is suitable for high density growth of fissiparism chytrid and increases the output of the DHA. The DHA comprises a carbon source, amino acids, lysine, inorganic salts, vitamins, trace elements and water. The carbon source is glucose, the amino acids comprise alanine, methionine, cysteine, lysine, histidine, glutamic acid, glutamine, isoleucine, threonine and tryptophan; the vitamins comprise VB1, VB12, biotin, lipoic acid and folic acid; the inorganic salts comprise NaCl, KCl, MgCl2, CaCl2, Na2HPO4 and KH2PO4; and the trace elements comprise AlCl3, H3BO3, ZnSO4, MnCl2, CuSO4, FeSO4 and (NH4)6Mo7O24.4H2O. The synthetic medium is utilized for culturing the fissiparism chytrid based on a shake flask, and the output of the DHA is 9.8g/L.