Diabetic nephropathy (DN) is one of the most serious and general complications of diabetes, while schedules for prevention and treatment are unsatisfactory. Leucine aminopeptidase (LAP) as one of the biomarkers of DN is associated with glomerular and/or tubular injury. Imaging of LAP activity in DN disease model in vivo is thus beneficial for early diagnosis and prevention of DN, but such a strategy is still lacking. Herein, an enzyme-activated probe HD-LAP with a NIR fluorescence emission for specific detection of LAP activity in the DN model is designed and synthesized. HD-LAP has a significant fluorescence enhancement after reacted with LAP and shows a NIR fluorescence emission at 704 nm based on intramolecular charge transfer mechanism. Moreover, HD-LAP can be employed to image LAP activity in HK-2 and HepG2 cells. More importantly, HD-LAP is the first example to real-time image LAP in DN mice and clinical serum samples. These results demonstrated that HD-LAP is promising as a powerful tool for the research on LAP associated diabetic diseases in future.