The invention discloses a phellinus linteus mother strain tissue isolation transfer-tube culture method, mainly aiming at solving the problems of an existing phellinus linteus mother strain culture method which is too low in one-time inoculum size, slow in mycelium germination rate, high in pollution rate and the like. The culture method disclosed by the invention comprises the following steps: (1) cutting a massive tissue from a fruiting body of phellinus linteus, inoculating the massive tissue into a test-tube culture medium, and culturing in a constant-temperature darkroom until yellow villous mycelia appear; (2) when the mycelia are 2-3cm away from the massive tissue, picking the culture medium containing edge mycelia, transferring and culturing in another test-tube culture medium, and transferring for more than one time so as to obtain purified mycelia; and (3) as the test-tube culture medium is full of the purified mycelia, dicing the test-tube culture medium full of the purified mycelia, and inoculating and culturing the massive culture medium on an enlarged culture medium so as to obtain a phellinus linteus mother strain. The culture method disclosed by the invention has the advantages of effectively increasing the one-time inoculum size, reducing labor intensity, accelerating a germination rate, reducing a pollution rate and the like.