CD13/aminopeptidase N (APN), which is a zinc-dependent metalloproteinase, plays a vital role in the growth, migration, angiogenesis, and metastasis of tumours. Thus, in situ molecular imaging of endogenous APN levels is considerably significant for investigating APN and its different functions. In this study, a novel two-photon near-infrared (NIR) fluorescence probe DCM-APN was prepared to perform in vitro and in vivo tracking of APN. The N-terminal alanyl site of probe DCM-APN was accurately hydrolysed to the amino group, thereby liberating strong fluorescence owing to the recovery of the Intramolecular Charge Transfer (ICT) effect. By considering its outstanding selectivity, ultra-sensitivity (DL 0.25 ng mL-1) and favourable biocompatibility, the probe DCM-APN was used to distinguish between normal cells (LO2 cells) and cancer cells (HepG-2 and B16/BL6 cells). Furthermore, migration of hepatocellular carcinoma cells was apparently inhibited by ensuring that the APN catalytic cavity was occupied by bestatin. The identification of three-dimensional (3D) fluorescence in cancer tissues was completed under two-photon excitation coupled with lighting up hepatocellular carcinoma tumours in situ; this revealed that probe DCM-APN is an effective tool for detecting APN, thereby assisting in the early diagnosis of tumour in clinical medicine.