Specific cancer diagnosis at an early stage plays a significant role in preventing cancer metastasis and reducing cancer mortality. Thus, exploring specific and sensitive fluorescent probes to realize early cancer diagnosis is an urgent need in clinic. Aminopeptidase N (APN/CD13), overexpressed in numerous malignant tumors, is an important tumor biomarker associated with cancer progression, invasion, and metastasis. In this study, a novel fluorescent molecule APN-SUB, capable of monitoring APN in real time, is encapsulated in a pH-responsive block copolymer (termed APN-SUB nanoprobe) for cancer diagnosis. APN-SUB contains a fluorophore center and a trigger moiety (leucine group), which is covalently conjugated on the fluorophore with an amide bond. The hydrolysis of the amide bond in APN-SUB activated by APN leads to a red shift of maximum fluorescence emission wavelength from 495 nm to 600 nm, realizing dual-color transformation from green to red. Moreover, the APN-SUB nanoprobe with pH-responsiveness is prepared to improve the accumulation and the release rate in the tumor region. It is worth noting that the APN-SUB nanoprobe exhibits good performance for APN imaging, namely, superior limit of detection (0.14 nU mL-1), excellent selectivity and strong photostability. More importantly, the APN-SUB nanoprobe can be successfully employed as a color-convertible fluorescent probe for cancer diagnosis by tracking the activity of APN with high specificity and sensitivity in vivo, demonstrating its potential value for cancer diagnosis.