The invention discloses a culture medium and a culture method of a pleural mesothelioma organ. The culture medium comprises a basic culture medium Advanced DMEM / F12 and a specific addition factor, the specific addition factor is prepared from the following components: B27 which does not contain vitamin A, N2, N-acetylcysteine, EGF (Epidermal Growth Factor), Noggin, R-spondin 1, Wnt3a, CHIR99021, Thiazovivin, VEGFR (Vascular Endothelial Growth Factor Receptor), PDGFR, Penicillin and Streptomycin mixed liquor and Primocin. The culture medium contains the least components required by pleural mesothelioma organoid culture, can effectively culture mesothelial cell-derived tumor tissues, does not contain FBS, saves the cost, and reduces the cytotoxicity and inhibitors caused by FBS at the same time. The pleural mesothelioma organoid cultured by the invention maintains the morphological structure and gene characteristics of the primary tissue.