In the current paper, three activity-based colorimetric and ratiometric fluorescent probes based on a naphthalimide fluorophore were well designed and synthesized, which can be recognized and hydrolyzed by aminopeptidase N (APN) at both the enzymatic and cellular level by following the fluorescent emission wavelength change from blue to green light. As a result, these molecules were successfully identified as the first ratiometric fluorescent probes for APN cell imaging.