Herein we present the genetic encoding of 7-aminocoumarin-based lysine derivatives, ACouK and AFCouK, into proteins in both bacterial and mammalian cells and the characterization of FRET pairs comprising ACouK or AFCouK as the donor and GFP as the acceptor. We further report the application of the FRET pairs to construct fully genetically encoded ratiometric probes for detecting deubiquitinases and screening for inhibitors.