As a detection instrument developed rapidly in recent years, the spectral flow cytometer primarily complemented traditional flow cytometry. This article demonstrated the performance of three spectral flow cytometers in the detection of fluorescent materials, demonstrating their potential applications in the field of biological materials. Studies showed that the spectral flow cytometer could not only explore fluorescence from non-primary bands of known fluorescent dyes, but also decompose overlapping spectra between fluorescent materials, and reagent kit detection channels, resulting in more accurate analytical results. Additionally, it could detect the spectral shifts of fluorescent nanomaterials within cells. In all, the spectral flow cytometer could record the spectral characteristics of fluorescent materials entering cells, analyze the spectral behavior of fluorophores within cells, and spectrally resolve fluorescence signals that overlapped with detection channels, thus providing objective fluorescence data. These capabilities opened up new paths for the research of fluorescent materials, providing theoretical foundations as well as practical references for the widespread use of spectral flow cytometry in biomaterials science.