K27-linkage poly-ubiquitination plays important roles in DNA damage repair and autoimmunity. Identification of K27-linkage targeting deubiquitinases (DUBs) is essential for understanding their regulatory mechanisms. Here we report an aryl-azide-based photoaffinity diubiquitin (diUb) probe for profiling K27-linkage targeting DUBs. This probe shows high selectivity and sensitivity towards K27-linkage DUBs in vitro. It can also be used in cell lysate for identifying and probing K27-linkage targeting DUBs in real proteomes. Our work suggests that the photoaffinity-based strategy may provide a new useful approach for the construction of other isopeptide linkage targeting DUB probes.