This chapter presents advances in aptamer selection technologies and the main formats of biosensors, which were frequently employed in aptasensor development. It discusses the state of aptamers, as applied to medical diagnosis for specifically cancer and diagnosis or pathogenic microorganisms. The chapter also presents an overview of aptamer–nanomaterial conjugates in many applications such as diagnosis, bioimaging, and theranostics. Diseases like cancer originate from genomic mutations that can cause changes of cells at morphological and molecular levels. Exploring nucleic acids for ligand-binding molecules resulted in the discovery of aptamers in 1990s, where their novel properties were exploited for the development of new analytical formats. Bioconjugation is a chemical strategy for covalent modification of biomolecules. A noninvasive aptamer imaging method based on stem-loop- or linear-structured DNA molecular beacon was developed to determine endogenous adenosine triphosphate concentrations at high spatiotemporal resolution. Several techniques have been used for fabricating electrochemical aptasensors, such as electrochemical impedance spectroscopy, potentiometry, and amperometry.