Post-translational modification of proteins by Ubiquitin (Ub) and Ubiquitin-like proteins (Ubls) can be reversed by deconjugating enzymes, which have been implicated in different pathways and associated with various human diseases. To understand the activity and dynamics of deconjugating enzymes, multiple synthetic and semi-synthetic Ub/Ubl probes have been developed, and some of them have been applied to screen inhibitors of deconjugating enzymes. Since these Ub/Ubl probes are generally not cell-permeable, different strategies have been developed to deliver Ub/Ubl probes to live cells. However, till now no Ub/Ubl probes can be expressed in live cells to directly report on the activities of deconjugating enzymes in the most relevant cellular environment. Here, we genetically encoded cross-linkable Ub/Ubl probes in live E. coli and HEK293T cells. These probes can cross-link with deconjugating enzymes in vitro and in vivo, and the extent of cross-linking is dependent on the activities of deconjugating enzymes.