The invention discloses an improved Neurobasal B27 culture medium. One liter of a Neurobasal culture solution comprises 0.09-0.11 g of biotin, 18-22 micrograms of corticosterone, 1-3 g of L-carnitine, 0.9-1.1 g of linoleic acid, 0.9-1.1 g of ethanol amine, 0.9-1.1 g of linolenic acid, 13-17 g of D(+)-galactose, 6-7 micrograms of progesterone, 15-17 g of putrescine dihydrochloride, 0.08-0.12 g of retinyl acetate, 2.2-2.8 g of thyroid-hormone-removing bovine serum albumin, 0.8-1.5 g of DL-alpha-tocopherol, 2-3 g of catalase, 0.9-1.2 g of DL-alpha-tocopheryl acetate, 0.8-1.3 g of reduced glutathione, 42-52 micrograms of lipoic acid, 2-3 g of superoxide dismutase, 4-6 g of transferring, 3-5 g of human insulin and 0.12-0.16 mg of sodium selenite. The invention further discloses a preparing method and application of the improved Neurobasal B27 culture medium. The improved Neurobasal B27 culture medium can be used for researching the influence of thyroid hormones on the process that embryonic stem cells are differentiated into nerve cells.