The invention relates to a protein-free, hydrolysate-free and serum-free culture medium and a preparation method thereof. The culture medium contains the following substances: manganese gluconate, calcium citrate malate, disodium edetate, progesterone, alanyl-glutamine,glycerophosphate, fructose, vitamin C, vitamin E, sodium selenite, hydroxypropyl-beta-cyclodextrin, cholesterol, malic acid, oxaloacetic acid, vitamin B12, lipoic acid, ferrous sulfate, ferric citrate, uric acid, taurine, reduced glutathione, zinc sulfate, copper sulfate and ferric nitrate. The protein-free, hydrolysate-free and serum-free culture medium has the advantages that the cost is low, the difference among different batches of products is small, the purification step of down-stream products is simplified, and potential safety hazards are reduced.