Development of nanoparticles for super-resolution imaging (sriNPs) can greatly enrich the toolbox of robust optical probes for biological studies. Moreover, sriNPs enable us to monitor the behavior of engineered nanomaterials in complex biological environments with high spatial resolution, which is important for advancing our understanding of nano–bio interactions. Up to now, reports on sriNPs have been scarce. In this work, we report a facile strategy to prepare protein-based fluorescent NPs that can be utilized as probes in super-resolution microscopy. The method is simple and straightforward, and easily extendible to other types of fluorophores. By using Atto647N–transferrin NPs as an example, we have achieved a roughly four-fold resolution improvement by using STED nanoscopy. These protein-based sriNPs possess excellent biocompatibility, good colloidal stability and photostability, making them attractive candidates for biological studies. Moreover, STED nanoscopy enables the precise imaging of NP structures in living cells, and revealed the co-existence of multiple NPs within one endosomal vesicle.