Recent studies revealed that heterogeneity is a general phenomenon among microbes with identical genomes, and it is closely related to physiological functions of microbes. The multiple heterogeneous states of microbes requires single-cell level tools to uncover. Flow cytometry is one of the most important methods to obtain accurate information on quantitative distribution of heterogeneous states of microbes. However, the small size, much less biomolecule contents and general lack of specific reagents limit the application of conventional flow cytometry to investigations of the heterogeneity of microbes. In this study, we applied a newly developed flow cytometer with increased resolution and sensitivity on three label-free parameters which include FSC,SSC and autofluorescence level of NAD(P)H to analyse Bacillus subtilis. And for the first time we revealed that the bacterial culture exhibited complicated and dynamically distributed multiple heterogeneous subgroups depended on its growth stage. This method has low background noise, high sensitivity and great resolution. The newly identified multiple subpopulations of Bacillus subtilis and their changes dynamically associated with the biological functions are likely to provide new opportunities to understand physiological change patterns and their underlying molecule mechanisms. We also discussed that the new method based on flow cytometric measurements of non-label parameters with high sensitivity and resolution had great potency for studies on the heterogeneity of various microbes.