Spatiotemporally manipulating the in situ immobilization of theranostic agents within cancer cells to improve their bioavailability is highly significant yet challenging in tumor diagnosis and treatment. Herein, as a proof-of concept, we for the first time report a tumor-targetable near-infrared (NIR) probe DACF with photoaffinity crosslinking characteristics for enhanced tumor imaging and therapeutic applications. This probe possesses great tumor-targeting capability, intensive NIR/photoacoustic (PA) signals, and a predominant photothermal effect, allowing for sensitive imaging and effective photothermal therapy (PTT) of tumors. Most notably, upon 405 nm laser illumination, DACF could be covalently immobilized within tumor cells through a photocrosslinking reaction between photolabile diazirine groups and surrounding biomolecules resulting in enhanced tumor accumulation and prolonged retention simultaneously, which significantly facilitates the imaging and PTT efficacy of tumor in vivo. We therefore believe that our current approach would provide a new insight for achieving precise cancer theranostics.