The invention relates to a human primary tumor cell separation preparation method which comprises the following steps of washing tumor cells which are separated out by using a 0.9 percent sodium chloride injection, removing blood, and shearing necrotic and paeoniae tissues; shearing the tumor tissues into 1mm<3>, placing the shorn tumor tissues in a centrifugal tube, adding type I collagenase in the centrifugal tube, carrying out cold digestion overnight, adding hyaluronidase and DNA enzyme next day, carrying out digestion, sieving a mixed liquid after digestion by a cell screen, collecting a filtrate, and carrying out centrifugation to remove supernatant to obtain a single-cell suspension; adding a serum-free primary culture medium, beating, uniformly mixing, counting cells, inoculating the cells into a culture bottle, supplementing the serum-free primary culture medium, and placing the culture bottle in a carbon dioxide constant-temperature and constant-humidity incubator to carry out culture; and carrying out cryopreservation. The human primary tumor cell separation preparation method provided by the invention is high in harvest rate of prepared cells, enables the cells to be rapid in proliferation, can improve the proliferation capability of human primary tumor cells, can increase the proliferation speed of the human primary tumor cells, reduces the culture period and has prominent progress.