Selective fluorescence imaging of analytes is a challenge for monitoring diseases as homologues interfere with the imaging agents. Leucine aminopeptidase (LAP), a kind of protease, is related to tumor pathogenesis. The known LAP fluorescent probes based on leucine recognition have limited selectivity. Herein, a selective t-butyl-alanine recognition unit for LAP through the ligand regulation strategy is prepared as a new near-infrared (NIR) fluorescent probe (DCM-LAP) having a large Stokes shift of 214 nm and a high sensitivity with a detection limit of 168 mU/L. DCM-LAP has an enhanced response toward LAP with NIR fluorescence at 656 nm based on intramolecular charge transfer. The probe is selective without being interfered with by biological enzymes including the aminopeptidase N (APN). DCM-LAP can image LAP activity in living cells. It can also visualize the cell invasion and migration processes. DCM-LAP is employed in the real-time imaging of LAP in tumor-bearing nude mice and guides in the accurate resection of breast tumors. It also distinguishes tumor tissues from normal with a high tumor-to-normal ratio (9.8). The DCM-LAP probe can thus assist in the investigations of LAP-associated clinical disease.