Objective The successful establishment of a tumor cell bank is based on the premise that the target cells can be cultured by a legitimate approach. In this experiment, we used primary culture to select and detect breast cancer cells in vitro, which can provide experimental ideas and methods for the establishment of a living tumor tissue cell bank.Methods Fifty-two specimens were collected over a two-year period from people with breast cancer who needed surgical treatment in our hospital. Cells were isolated and used to establish successful cell culture. Cell activity and cell purity were measured before liquid nitrogen cryopreservation. Results (1) At the initial culture stage, cells grew with adherence. Cell multiplication could be seen after the cell medium was exchanged three times. Cell viability was above 86％, while the viability of the target cells was above 75％, as detected by hematoxylin and eosin (HE) staining. (2) The number of breast cancer cells decreased, while the number of fibroblasts increased after five rounds of passage. (3) The success rate was 73.08％, which did not include polluted cells and those that were not successfully cryopreserved. Conclusion (1) breast cancer cells could be selected from primary culture in vitro through an appropriate method. (2) Exchange of the cell medium and further cell passage improved cell multiplication. (3) The experimental results could be monitored using trypan blue and HE staining. (4) The success of breast cancer cell culture in vitro could be used as a reference for other cell culture, so as to establish a tumor tissue cell bank.