Photocatalysis is a cost-effective approach to producing renewable energy. A thorough comprehension of carrier separation at the micronano level is crucial for enhancing the photochemical conversion capabilities of photocatalysts. However, the heterogeneity of photocatalyst nanoparticles and complex charge migration processes limit the profound understanding of photocatalytic reaction mechanisms. By establishing the precise interrelationship between microscopic properties and photophysical behaviors of photocatalysts, single-particle fluorescence spectroscopy can elucidate the carrier separation and catalytic mechanism of the photocatalysts in situ, which provides perspectives for improving the photocatalytic efficiency. This Review primarily focuses on the basic principles and advantages of single-particle fluorescence spectroscopy and its progress in the study of plasmonic and semiconductor photocatalysis, especially emphasizing its importance in understanding the charge separation and photocatalytic reaction mechanism, which offers scientific guidance for designing efficient photocatalytic systems. Finally, we summarize and forecast the future development prospects of single-particle fluorescence spectroscopy technology, especially the insights into its technological upgrading.