Tumor-educated platelets (TEPs) have been widely reported to have promising application potential; nonetheless, platelet isolation from peripheral blood is an important but neglected step in TEPs research for platelet-based liquid biopsy. In this article, we discussed some common influence factors for platelet isolation. To investigate the factors involved in platelet isolation, a prospective multicenter study was conducted on healthy Han Chinese adults (18 to 79 years of age). A total of 208 individuals were included in the final statistical analysis out of the 226 healthy volunteers who were prospectively enrolled from four hospitals. The primary study metric was the platelet recovery rate (PRR). The similar pattern was observed in the four hospitals, The PRR at room temperature (23 degrees C +/- 2 degrees C) was slightly higher than the PRR at cold temperature (4 degrees C +/- 2 degrees C). Moreover, the PRR gradually decreased as the storage time increased. The PRR for samples within 2 hours of storage is significantly higher than for samples beyond 2 hours (p < .05). Additionally, PRR was also affected by the equipment used in different centers. This study confirmed several factors that influence platelet isolation. In our study, we indicated that platelet isolation should be performed within two hours of peripheral blood draw and held at room temperature until isolation, and that centrifuge models should be fixed during the extraction process, which will further improve the research progress of platelet-based liquid biopsy in cancer.