We report a genetically encoded, residue-selective protein photo-crosslinker enabling covalently crosslinking protein-protein interactions with proximal lysine upon UV light activation in vitro and in living cells. Combining the advantages of temporal control and residue selectivity, a new crosslinking approach was developed, producing predictable crosslinking site and crosslinked peptides, and capturing elusive enzyme-substrate interaction with directly interacting lysine. This strategy represents higher spatiotemporal resolution and reliability for investigating protein-protein interactions, thus, better understanding of biological processes and pathophysiology.