The invention provides a method and culture medium for in-vitro isolated culture of glioma stem cells. The method comprises the following steps of 1, adding cell tumor tissues into a PBS buffer solution, removing blood clots and necrotic cells, and cutting the treated cell tumor tissues into paste; 2, adding an Accutase solution to digest tissue blocks into a cell suspension; 3, adding a GC gliomacell culture medium, performing uniform mixing, stopping digestion, and filtering the cell suspension through a filter screen (BD) to obtain a single-cell suspension; 4, adding PBS, performing re-suspending precipitation, washing the cells, and removing an enzyme solution; 5, adding an erythrocyte lysis solution to lyse erythrocytes, discarding a supernatant, and adding PBS to wash cell precipitates in order to remove the erythrocyte lysis solution; and 6, adding an NXFS glioma stem cell culture medium to a Laminin-coated 6-hole cell plate, and carrying out primary culture on the glioma stemcells. The glioma stem cells with the purity of about 80% can be obtained.