BACKGROUND: Low-temperature cryopreservation is a common method for scientific research and clinical sample preservation when utilizing flow cytometry. In flow cytometry data analysis, traditional manual "gating" is susceptible to past experience and faces the challenge of manual subjective bias, time-consuming, and multidimensional data analysis. With the development of algorithms, the advantages of dimensionality reduction and clustering in result analysis are gradually becoming more prominent. METHODS: Flow cytometry was used to detect the effects of cryopreservation and freeze-thaw cycle on T-cell subsets, and to analyze the data using automated algorithmic analysis and conventional manual "gating" methods. RESULTS: The results showed that the number and viability of cells decreased slightly after one freeze-thaw within 2 weeks of cryopreservation, and there was no significant change in the subpopulation proportions and spatial locations by both analysis methods. The changes were significant with the increase of cryopreservation time and freeze-thaw cycle, which may be due to changes in the molecular conformation of the maker as a result of cryopreservation. CONCLUSION: The results indicate that both analysis methods have reached similar conclusions, but the repeatability and objectivity of automated algorithmic analysis have compensated for the uncertainty brought about by the subjective discretization of traditional manual "gating." In addition, the automated algorithmic analysis more intuitively highlights the spatial positional variations in the relationships between cell populations.