Cancer is still one of the world’s foremost killers; the methods of its detection are very important to be developed in the early stages and as accurately as possible. Aptamers, which are single-stranded nucleic acid entities, have demonstrated substantial potential as diagnostic instruments for cancer owing to their pronounced affinity and specificity for designated targets. Due to their unique properties, such as chemical stability, low immunogenicity, ease of synthesis, and tunable functionality, they are suitable candidates as alternatives to traditional antibodies. The key is an advancement in synthesizing high-affinity aptamers specifically for different cancer biomarkers that were developed using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique. New advances in bioengineering and nanotechnology have increased the potential of aptamers as diagnostic tools. By integrating aptamers with nanomaterials, researchers have developed highly specific and sensitive biosensors capable of detecting low concentrations of cancer biomarkers. In addition, the application of artificial intelligence (AI) in aptamer design is propelling the search for new candidates and improving their diagnostic performance. Despite these advances, challenges to clinical translation remain in terms of off-target effects, stability in complex biological environments, and scalability. This chapter provides a detailed overview of the current state of the art of aptamer-based cancer diagnostics on a mechanistic, novel, and applicative basis. It discusses the challenges to the development and commercialization of biosensor technologies and highlights the potential for aptamers to be incorporated into them. In the last section, the significance of aptamers in enhancing early cancer diagnosis and personalized medication is emphasized, along with potential research and innovation pathways for the future.