Nanoparticles (NPs) show great potentials in biomedicine and therapeutics fields with the specific physical and chemical properties. As the physiological functions and toxicity of NPs depend heavily on their intracellular locations, the distribution of NPs within single cells presents to be of vital importance. Herein, a nano laser probe (NLP)-based mass spectrometry imaging (MSI) technique was utilized for visualization of titanium dioxide nanoparticles (TiO 2 NPs) within single cells with an imaging pixel size of 500 nm. Using a micro-lensed fiber for laser introduction, the sampling crater diameter of about 420 nm was obtained. In addition, images of all titanium isotopes and their oxides were acquired for the same cells. The MSI results indicate that TiO 2 NPs simply accumulate in lysosomes, which is in accordance with the results of laser scanning confocal microscopy (LSCM). With a great feasibility and reliability, this technique has great potential for the research of NPs in biomedicine and clinical fields.