The invention discloses a Peziza repanda Pers. strain separation method which is characterized by comprising the following steps: preparing a culture medium, respectively pouring the culture medium into culture bottles, capping, autoclaving, cooling, carrying out preculture in a culture chamber for 3 days, and carrying out material inoculation if no infectious microbe pollution occurs; flushing the material with tap water, sterilizing, absorbing water with sterile filter paper, cutting the required position, taking a 5mm block, inoculating the material to the obtained culture bottles, and culturing; putting the culture bottles in a culture chamber with the temperature of 25 DEG C or so, and culturing for 7-10 days to form the callus; and dicing the formed callus, inoculating to a fresh PDA (potato dextrose agar) culture medium, putting the PDA culture medium in a constant-temperature environment of 25 DEG C or so, and culturing, wherein the hyphae can grow out after 3-5 days, and can be full of the test tube after 15 days. The method solves the problem that the Peziza repanda Pers. strain is difficult to obtain, and obtains the high-purity Peziza repanda Pers. strain.