Imaging and quantification of nanoparticles in single cells in their most natural condition are expected to facilitate the biotechnological applications of nanoparticles and allow for better assessment of their biosafety risks. However, current imaging modalities either require tedious sample preparation or only apply to nanoparticles with specific physicochemical characteristics. Here, the emerging hyperspectral stimulated Raman scattering (SRS) microscopy, as a label‐free and nondestructive imaging method, is used for the first time to investigate the subcellular distribution of nanoparticles in the protozoan Tetrahymena thermophila. The two frequently studied nanoparticles, polyacrylate‐coated α‐Fe2O3 and TiO2, are found to have different subcellular distribution pattern as a result of their dissimilar uptake routes. Significant uptake competition between these two types of nanoparticles is further discovered, which should be paid attention to in future bioapplications of nanoparticles. Overall, this study illustrates the great promise of hyperspectral SRS as an analytical imaging tool in nanobiotechnology and nanotoxicology. As a label‐free imaging technique, hyper‐spectral stimulated Raman scattering allows the determination of the distribution of dissimilar nanoparticles in single cells or unicellular organisms. Significant uptake competition between dissimilar nanoparticles is also revealed by this method, which should be considered not only in medical/biological applications but also in safety assessments of nanoparticles.