Supramolecular fluorescent hydrogels (SFH) have shown great potential as detecting probes for biomedical applications such as cell imaging, disease diagnosis, bio-sensing etc. This review aims to summarize the recent developments and trends in SFH, investigations of their preparation methods, fluorescent properties of SFH, their prospective applications and interactions involved in SFH based bio-imaging, along with the perspectives on future opportunities and the remaining challenges confronting this research field. After a brief introduction, the categories of SFH, such as fluorescent gelators (self-assembly) and dyes diffused into the matrix of gelators (co-assembly), are illustrated. Various possible uses of SFH as a bio-imaging tool have been realized by their non-covalent interactions like aromatic–aromatic interactions, hydrogen bonding, and hydrophobic interactions etc. More importantly, the uses of SFH in helping address major questions about the consequences of the fluorescent molecules, comprising enzymes and bola-amphiphiles, are also described. This review will serve as a rationalized introduction and reference for researchers who are interested in exploring SFH as bio-imaging probes and will motivate new designs along with instigation of persistent efforts in this hot subject area with great prospects.