Hydrogels are excellent water-swollen polymeric materials for use in wearable, implantable, and disposable biosensors. Hydrogels have unique properties such as low cost, ease of preparation, transparency, rapid response to external conditions, biocompatibility and self-adhesion to the skin, flexibility, and strain sensitivity, making them ideal for use in biosensor platforms. This review provides a detailed overview of advanced applications of stimuli-responsive hydrogels in biosensor platforms, from hydrogel synthesis and functionalization for bioreceptor immobilization to several important diagnostic applications. Emphasis is placed on recent advances in the fabrication of ultrasensitive fluorescent and electrically conductive hydrogels and their applications in wearable, implantable, and disposable biosensors for quantitative measurements. Design, modification, and assembly techniques of fluorescent, ionically conductive, and electrically conductive hydrogels to improve performance will be addressed. The advantages and performance improvements of immobilizing bioreceptors (e.g., antibodies, enzymes, and aptamers), and incorporating fluorescent and electrically conductive nanomaterials are described, as are their limitations. Potential applications of hydrogels in implantable, wearable, disposable portable biosensors for quantitative detection of the various bioanalytes (ions, molecules, drugs, proteins, and biomarkers) are discussed. Finally, the global market for hydrogel-based biosensors and future challenges and prospects are discussed in detail.