Cancer represents one of the most complex and challenging diseases of our time, characterized by dynamic molecular landscapes and intricate cellular mechanisms. This chapter provides a comprehensive exploration of cancer biology, emphasizing aspects crucial for developing next-generation diagnostic and therapeutic approaches. Beginning with fundamental concepts of cellular transformation, we examine the hallmarks of cancer that distinguish malignant cells from their normal counterparts. The chapter provides a brief overview of the molecular mechanism of cancer, including genetic alterations, signaling pathways, tumor microenvironment (TME), tumor heterogeneity, and their clinical implications in cancer diagnosis, and the limitations of current detection methods are also discussed. The chapter introduces cancer biomarkers, including both traditional protein biomarkers and emerging molecular biomarkers, and discusses the issues associated with biomarker detection and utilization that may pose additional challenges in cancer diagnosis, thus highlighting the deficiencies in current diagnostic methods. This chapter lays the groundwork for the introduction of aptamer-based biosensing systems as a more promising technology for cancer detection and monitoring, thereby demonstrating how such systems can address the shortcomings of existing diagnostic methods.