Stimuli-responsive supramolecular assemblies of aggregation-induced emission (AIE) materials have revolutionized biosensing by offering superior sensitivity, selectivity, and adaptability. Unlike conventional fluorophores, AIE luminogens (AIEgens) exhibit enhanced fluorescence upon aggregation, overcoming aggregation-caused quenching (ACQ) and enabling dynamic fluorescence modulation in complex biological environments. This chapter explores the integration of AIE materials with supramolecular strategies to develop highly responsive biosensors for detecting small molecules, proteins, enzymes, nucleic acids, and pathogens. These supramolecular assemblies leverage noncovalent interactions such as electrostatic forces, π–π stacking, and host–guest chemistry to enable fluorescence “turn-on” or “turn-off” responses upon exposure to molecular stimuli, including pH shifts, enzymatic activity, and biomolecular recognition events. The synergy between AIE materials and supramolecular architectures has led to breakthroughs in ATP detection, amino acid sensing, protease and phosphatase monitoring, and disease biomarker identification. Additionally, recent advances in metal nanocluster-supported AIE sensors and polymer-based AIE biosensors have enhanced their stability and biocompatibility, paving the way for real-time, high-precision diagnostics. By bridging fundamental research with practical applications in medical diagnostics, environmental monitoring, and food safety, this chapter highlights the transformative potential of AIE-based supramolecular biosensing platforms in next-generation analytical and biomedical sciences.