Cell-permeable ubiquitin (Ub) probes labeled with a fluorescent or biotin tag have become effective tools for studying the activity of deubiquitinating enzymes (DUBs) in living cells. However, such probes are usually obtained through total chemical synthesis. In this study, we develop a Sortase A (SrtA)-mediated semi-synthetic strategy for preparing cell-permeable Ub-propargylamide (Ub-PA) with a fluorescent or biotin tag. We demonstrated efficient internalization of the fluorescent Ub-PA probe by live-cell fluorescence confocal microscopy. Additionally, the probe enables selective labeling of distinct DUB families through its attached biotin tag, facilitating subsequent proteomic profiling.