A bicyclo[6.1.0]nonyne (BCN)-based cyclooctyne reagent bearing a photocrosslinking diazirine (DAz) group and a biotin affinity handle, BCN–DAz–Biotin, is reported. BCN–DAz–Biotin is capable of simultaneously delivering photocrosslinking and affinity tags to azide-labeled biomolecules, enabling photoactivated capture and enrichment/detection of interacting species in native contexts.