The invention provides a separation preparation method for human primary cancer cells. According to the separation preparation method, the culture process is divided into two periods, culture is carried out with culture mediums with fetal calf serum of different concentrations respectively, the concentrations of the fetal calf serum in the culture mediums are increased gradually, then the adaptability and the activity of primary cancer cells are improved gradually, relatively rapid cell proliferation and relatively high yield are achieved, and meanwhile good hereditary stability is achieved; due to addition of sodium pyruvate, MaxCitro, Y-27632 and griseofulvin, bacterial and fungal infection can be prevented, cell proliferation can be promoted, and cell differentiation can be inhibited; and due to addition of a plant source recombinant human serum albumin in a cryoprotectant, a part of fetal calf serum can be replaced, the use amount of the fetal calf serum can be reduced, and thus the cost can be lowered on premise that the effect is not affected.