Understanding virus-host interactions is crucial for vaccine development. This study investigates such interactions using fluorescent nanodiamonds (FNDs) coated with vaccinia envelope proteins as the model system. To achieve this goal, we noncovalently conjugated 100 nm FNDs with rA27(aa 21-84), a recombinant envelope protein of vaccinia virus, for glycosaminoglycans (GAGs)-specific targeting and imaging of living cells. Another recombinant protein rDA27(aa 33-84) that removes the GAGs-binding sequences was also used for comparison. Three types of A27-coated FNDs were generated, including rA27(aa 21-84)-FND, rDA27(aa 33-84)-FND, and hybrid rA27(aa 21-84)/rDA27(aa 33-84)-FND. The specificity of these viral protein-FND conjugates toward GAGs binding was examined by flow cytometry, fluorescence microscopy, and gel electrophoresis. Results obtained for normal and GAGs-deficient cells showed that the recombinant proteins maintain their GAG-targeting activities even after immobilization on the FND surface. Our studies provide a new nanoparticle-based platform not only to target specific cell types but also to track the fates of these immobilized viral proteins in targeted cells as well as to isolate and enrich GAGs-associated proteins on cell membrane.