A primary cell culture medium for gastric cancer and/or gallbladder cancer and cholangiocarcinoma, is composed of antibiotic-antimycotic, HEPES, GlutaMax, non-essential amino acids, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein FGF-10, human recombinant protein Wnt-3a, human recombinant protein Noggin, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, Nicotinamide, N-2 Supplement, cortisol, B27, ITS-X, Gastrin 1, Y-27632 and Advanced DMEM/F12 culture medium; wherein, the final concentration of penicillin in the antibiotic-antimycotic is 100-200U/mL; the final concentration of streptomycin in the antibiotic-antimycotic is 100-200pg/mL; the final concentration of amphotericin B in the antibiotic-antimycotic is 250-250ng/mL; the final concentration of the HEPES is 8-12mM; the final concentration of the GlutaMax is 0.8-1.2 % (volume percentage); the concentration of glycine in the non-essential amino acids is 80-120μM; the concentration of L-alanine in the non-essential amino acids is 80-120μM; the concentration of L-asparagine in the non-essential amino acids is 80-120μM; the concentration of L-aspartic acid in the non-essential amino acids is 80-120μM; the concentration of L-glutamic acid in the non-essential amino acids is 80-120μM; the concentration of L-proline in the non-essential amino acids is 80-120μM; the concentration of L-serine in the non-essential amino acids is 80-120μM; the final concentration of the human recombinant protein EGF is 10-100 ng/mL; the final concentration of the human recombinant protein bFGF is 10-50 ng/mL; the final concentration of the human recombinant protein HGF is 5-25 ng/mL; the final concentration of the human recombinant protein FGF-10 is 5-25 ng/mL; the final concentration of the human recombinant protein Wnt-3a is 200-300 ng/mL; the final concentration of the human recombinant protein Noggin is 100-200 ng/mL; the final concentration of the SB202190 is 5-10μM; the final concentration of the A83-01 is 0.25-1.25μM; the final concentration of Primocin is 1 % (volume percentage); the final concentration of the N-acetyl-L-cysteine is 0.5-2mM; the final concentration of the Nicotinamide is 5-10mM; the final concentration of the N-2 Supplement is 1 % (volume percentage); the final concentration of the cortisol is 20-50 ng/mL; the final concentration of the B27 is 1.5-2.5 % (volume percentage); the final concentration of the ITS-X is 0.8-1.2 % (volume percentage); the final concentration of the Gastrin 1 is 8-12nM; the final concentration of the Y-27632 is 5-20 μM; and the rest is the Advanced DMEM/F12 medium, wherein,: the gastric cancer is primary gastric cancer; and the gallbladder cancer and cholangiocarcinoma is primary gallbladder cancer and cholangiocarcinoma; or, the gastric cancer is a metastatic lesion of gastric cancer; and the gallbladder cancer and cholangiocarcinoma is a metastatic lesion of gallbladder cancer and cholangiocarcinoma. the gastric cancer is primary gastric cancer; and the gallbladder cancer and cholangiocarcinoma is primary gallbladder cancer and cholangiocarcinoma; or, the gastric cancer is a metastatic lesion of gastric cancer; and the gallbladder cancer and cholangiocarcinoma is a metastatic lesion of gallbladder cancer and cholangiocarcinoma.