Live cell imaging brings us into a new era of direct visualization of biological processes and molecular dynamics in real time. To visualize dynamic cellular processes and virus-host interactions, fluorescent labeling of proteins of interest is often necessary. Fluorescent proteins are widely used for protein imaging, but they have some intrinsic deficiencies such as big size, photobleaching, and spectrum restriction. Thus, a variety of labeling strategies have been established and continuously developed. To protect the natural biological function(s) of the protein of interest, especially in viral life cycle, in vivo labeling requires smaller-sized tags, more specificity, and lower cytotoxicity. Here, we briefly summarized the principles, development, and their applications mainly in the virology field of three strategies for fluorescent labeling of proteins of interest including self-labeling enzyme derivatives, stainable peptide tags, and non-canonical amino acid incorporation. These labeling techniques greatly expand the fluorescent labeling toolbox and provide new opportunities for imaging biological processes.