Electrochemical biosensors represent a rapidly growing field, offering sensitive and cost-effective detection of diverse biomarkers. However, achieving optimal performance often requires advanced materials engineering. Nanostructured materials (1–100 nm) have become integral to high-performance electrochemical biosensors due to their unique physicochemical properties. Their high surface-area-to-volume ratio enhances bioreceptor loading, while materials like carbon nanotubes and metallic nanoparticles facilitate rapid electron transfer, boosting sensitivity. Nanomaterials serve multiple crucial functions: signal amplification (e.g., AuNPs, carbon nanotubes, nanoporous metals, MXenes, dendrimers), improving stability and activity of bioreceptors, enhancing resistance to biofouling through surface modifications (e.g., PEGylation, zwitterionic polymers, hPG coatings), facilitating advanced synthetic bioreceptors, and acting as direct sensing elements. Notably, nanozymes (e.g., CeO₂, Fe₃O₄ NPs) mimic enzyme activity, while electrocatalytic nanomaterials (e.g., CuNPs, PtNPs, NiO) enable enzyme-free detection of analytes like glucose, creatinine, urea, and insulin. Despite challenges in reproducibility, long-term stability, biocompatibility, and device integration, ongoing research into rational design, hybrid nanomaterials, multiplexed detection, wearable/implantable sensors, and AI integration promises continued advancements. Nanomaterials have fundamentally transformed electrochemical biosensing, paving the way for next-generation diagnostic and monitoring tools with significant potential impacts.