Linkage-specific ubiquitin chain detection tools have emerged as effective approaches for profiling interactors of high-abundance ubiquitin chains (e.g., K48-, K6-, and K29-linked chains, accounting for >1% of total ubiquitin chains) in cellular processes. However, the low abundance of linear ubiquitin chains (<0.2% of total ubiquitin chains) impedes efficient dynamic capture of their interactors through direct application of linear ubiquitin chain-specific detection tools. In this study, we report the chemical synthesis of a cell-permeable, photoactivatable linear-diubiquitin-dehydroalanine (Dha) molecule via a structure-guided design strategy. This molecule enables time-resolved specific inhibition of OTULIN's deubiquitinase activity, thereby rapidly inducing the accumulation of endogenous linear ubiquitin chains in living cells. Coupled with linear ubiquitin linkage-specific detection tools, this approach facilitated comprehensive profiling of linear polyubiquitination interactors in both TNF-α-stimulated HeLa cells and macrophage colony-stimulating factor (M-CSF)-induced bone marrow-derived macrophages (BMDMs). Our work establishes a new paradigm for studying interactors of other low-abundance ubiquitin chains and underscores the central importance of chemical protein synthesis in developing advanced protein tools for biological research.