Metal nanoparticles released by human activities inevitably enter the aquatic environment. Numerous studies have shown that metal nanoparticles could induce reproductive toxicity and genetic toxicity in aquatic organisms. Furthermore, metal nanoparticles could be transmitted along the food chains which is a potential threat to human health. Quantitative analysis of intracellular metal nanoparticles is important for studying the biological effects of metal nanoparticles. In addition, there is heterogeneity among cells, and the individual cell with special physiological characteristics may influence or even determine the destiny of the cell population. However, most traditional methods for the quantitative determination of intracellular metal nanoparticles were based on the whole cell population whereas the heterogeneity of individual cells was neglected, which may result in the loss of significant information about cell populations that have important functions to the community. Therefore, the quantitative analysis of the intracellular metal nanoparticles in unicellular microorganisms, which locates at the bottom of the trophic level in the aquatic environment, is essential for understanding the interaction between metal nanoparticles and aquatic organisms and evaluating the potential risk of metal nanoparticles entering the food chain. In this review, we discuss the single-cell analysis methods for the quantitative determination of metal nanoparticles in aquatic unicellular organisms. The working principle and application of these methods and their pros and cons are summarized. This paper aims to provide a theoretical basis of the method selection for relevant studies in the future. Finally, we prospect the future research directions in this field according to the current research status.