The biomolecular recognition process based on the noncovalent interactions in aqueous solution exists in almost all living systems and plays important roles in catalytic reaction, material transport, information transmission, immune regulation, and so on, which is the unavoidable essence of life process. Studying biomolecular recognition in aqueous solution has been meaningful for understanding the nature of living systems with specific functions in the physiological environment, simulating biological processes, and developing new medical biomaterials. Among recent studies, water-soluble cationic fluorescent macrocycles are favored by researchers because of their rich binding sites, sensitive optical response, and good solubility in the aqueous environment. In this review, the development of biomolecular recognition by water-soluble cationic fluorescent macrocycles in the past decades has been comprehensively clarified. Firstly, the water-soluble cationic fluorescent macrocycles have been divided into pyridinium-based and imidazolium-based macrocycles according to their cationic moiety. Secondly, all the reported macrocycles have been summarized with their design strategies, photopsychical properties, and application for biomolecular recognition. Finally, based on the current research progress, we discuss the challenges faced in biomolecular recognition using water-soluble cationic fluorescent macrocycles and explore potential development in future.