Objective To investigate the effect of heat denaturation on peripheral blood cells analyzed by flow cytometry and to improve the application of Flow cytometry - Fluorescence in situ hybridization (Flow-FISH). Methods Heparinized peripheral blood were sampled from 5 patients without hematopoietic stem cell disease, isolated nucleated cells were labeled with CD45-Alexa Fluor®647, then denatured by high temperature. The scatter signals and fluorescence signals were analyzed by flow cytometry, on cells with and without heat denaturation separately. Results After heat denaturation, the sideward light scatter of granulocytes became markedly smaller, and the monocytes were difficult to be gated. The CD45 expression of all cells became weaker, especially the lymphocytes; and cell subgroups could be differentiated by CD45 and sideward light scatter, while it was not as clear as cells without heat denaturation. Conclusion Heat denaturation could change both light scatter signal and fluorescence intensity by flow cytometry, and the routine method to gate cell groups might be not precise. Therefore, fluorescence labeled lineage specific antibody may be useful method to apply in Flow-FISH.