Bioinspired polydopamine (PDA) has served as a universal coating to nanoparticles (NPs) for various biomedical applications. However, one remaining critical question is whether the PDA shell on NPs is stable in vivo. In this study, we modified gold nanoparticles (GNPs) with finely controlled PDA nanolayers to form uniform core/shell nanostructures (GNP@PDA). In vitro study showed that the PDA-coated GNPs had low cytotoxicity and could smoothly translocate to cancer cells. Transmission electron microscopy (TEM) analysis demonstrated that the PDA nanoshells were intact within cells after 24 h incubation. Notably, we found the GNP@PDA could partially escape from the endosomes/lysosomes to cytosol and locate close to the nucleus. Furthermore, we observed that the PDA-coated NPs have very different uptake behavior in two important organs of the liver and spleen: GNP@PDA in the liver were mainly uptaken by the Kupffer cells, while the GNP@PDA in the spleen were uptaken by a variety of cells. Importantly, we proved the PDA nanoshells were stable within cells of the liver and spleen for at least six weeks, and GNP@PDA did not show notable histological toxicity to main organs of mice in a long time. These results provided the direct evidence to support that the PDA surface modification can serve as an effective strategy to form ultrastable coatings on NPs in vivo, which can improve the intracellular delivery capacity and biocompatibility of NPs for biomedical application.