Fluorescent protein fusions with environmentally sensitive fluorophores have been widely used to investigate changes in the protein microenvironment. Unfortunately, these techniques often rely on bulky fluorescent proteins or tags to the N terminus or C terminus of the target protein, which can disrupt the behavior of the target protein and may limit their ability to investigate microenvironment changes with high spatial resolution. Here we develop a strategy to visualize microenvironment changes of protein substructures in real time by genetically incorporating environment-sensitive noncanonical amino acids (ncAAs) containing rotor-based fluorophores at specific positions of the target protein. Through computational redesign of aminoacyl-tRNA synthetase, we successfully incorporated these rotor-based ncAAs into several proteins in mammalian cells. Precise placement of these ncAAs at specific sites of proteins enables the detection of microenvironmental changes around individual residues during events such as aggregation, clustering, cluster dissociation and others.