Aptamer-based biosensors have emerged as a groundbreaking innovation in cancer diagnostics, offering unmatched sensitivity, specificity, and versatility. Aptamers, short single-stranded DNA (ssDNA) or RNA molecules, are engineered to bind selectively to cancer-related biomarkers, such as proteins, nucleic acids, or small molecules. Their unique ability to recognize targets with high affinity makes them an ideal component for biosensor development, enabling precise and early cancer detection. This chapter explores the principles and applications of aptamer-based biosensors in identifying tumor biomarkers, circulating tumor cells (CTCs), and genetic mutations associated with cancer. It emphasizes recent advancements in aptamer technology, including their integration into nanotechnology-enhanced platforms and electrochemical, optical, and fluorescence-based detection systems. These biosensors offer portability and real-time monitoring capabilities and pave the way for personalized and point-of-care (POC) diagnostics. The chapter further addresses the challenges in developing aptamer-based biosensors, including stability, scalability, and clinical validation, alongside strategies to overcome these limitations. The role of aptamers in multiplexed biosensors for the simultaneous detection of multiple biomarkers is highlighted, showcasing their potential in comprehensive cancer profiling. By leveraging their precision, low production cost, and adaptability, aptamer-based biosensors promise to transform cancer diagnostics and improve patient outcomes.