Fluorescence lifetime has significant applications in the field of fluorescence microscopy. Effective modulation of fluorescence lifetime can be achieved by controlling the radiative versus nonradiative processes of fluorophores. In this review, we systematically analyze and summarize chemical approaches that achieve fluorescence lifetime modulation for three different types of fluorophores, including small molecules, quantum dots, and metal complexes. In particular, this review is focused on the chemical mechanisms underlying fluorescence lifetime, the structure-function relationship that defines how chemical regulation is achieved, and the chemical principles that can be used to modulate different scaffolds of fluorophores. We aim to provide important resources for gaining a deeper understanding of fluorescence lifetime modulation, through in-depth investigation into the modulation mechanisms of various fluorescence systems. Perspectives are also proposed to enable future investigation on fluorescence lifetime modulation, a field that bears promises to drive the advancement and application of fluorescence imaging technology.