Fluorescent probes have long been valuable tools in the study of biological systems. With the ever-expanding range of known enzymatic biomarkers for disease, it has never been more important to develop synthetically facile, sensitive, selective, and robust methods for the detection of these analytes. The 1,8-naphthalimide fluorophore presents an ideal scaffold on which to design a range of fluorescent probes for an unknowable diversity of biomarkers. With tuneable photophysical properties, synthetic versatility, photostability and a large Stokes shift, the 1,8-naphthalimide has, and continues to be, exploited for the detection of a wide range of enzymatic conversions. This review will outline the recent progress towards the design and synthesis of 1,8-naphthalimide fluorophores for the detection of selected enzymatic conversions.