With the wide application of fullerenols in biomedicine, their environmental exposure risks and toxicity to organisms have been extensively studied. However, there is still a lack of knowledge about the distribution of fullerenols in organisms as an important aspect of their mechanism of toxicity. High-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is an emerging technology for researching the distribution of molecules in biological tissue samples. Using this high-resolution technique, we map the distribution of fullerenols in zebrafish tissues, and the results suggest that fullerenols enter the gill, intestine, and muscle tissues and even permeate the blood-brain barrier, reaching the brain of zebrafish after aquatic exposure. Moreover, from the MS images of fullerenols, the distribution amount of fullerenols is highest in the gill, followed by that in the intestine and the small amount in muscle and brain tissues. As an emerging environmental pollutant, the establishment of this research method will provide a new method for the study of the environmental toxicity of carbon nanomaterials. Our results also indicated that this high-resolution imaging method could be applied to explore the mechanism of interaction between carbon nanomaterials and biological systems at the cellular level in the future.