While linear ubiquitin plays critical roles in multiple cell signaling pathways, few substrates have been identified. Global profiling of linear ubiquitin substrates represents a significant challenge because of the low endogenous level of linear ubiquitination, the none-specific binding of linear polyubiquitin chain, and the high background interferences arising from heterogeneous ubiquitin linkages (e.g. K48-, K63-). We developed a bioorthogonal linear ubiquitin probe by site-specific encoding of a norbornene amino acid (NAEK) on ubiquitin. This probe facilitates covalent labeling of linear ubiquitin substrates in live cells and enables selective enrichment and identification of linear-ubiquitin modified proteins. we applied this probe in the profiling of substrates of linear ubiquitination in HEK293T cells and GSCs.