Abstract1,8‐Naphthalimide fluorophore, as the classical skeleton for fluorescent functional molecules, has gained much attention in the chemistry and biomedical field due to its excellent fluorescent properties, photostability, and structural flexibility. The rational design of the substituents on the C‐4 position and N‐position of 1,8‐naphthalimide chromophore can provide specific functions for the molecules, such as fluorescence sensing, energy‐trapping, and targeting. This review presents the design progress of 1,8‐naphthalimide‐based fluorescence sensors for metal ions and biological molecules, and 1,8‐naphthalimide‐functionalized photosensitive dyes in the biological field over the past years. We summarize the general fluorescent molecular design strategies, recent developments of 1,8‐naphthalimide in innovative photosensitizers, and the challenges, wishing to promote new ideas in developing smart and functional molecules from 1,8‐naphthalimide derivatives.