Nucleic acid aptamers are single-stranded DNA (ssDNA) or RNA oligonucleotides that have the ability to recognize different targets with their special secondary structures. Aptamers, which have high specificity and affinity, are gaining increasing importance in biosensor technologies as recognition elements that can be easily synthesized chemically and modified according to the need. The small size, stability, easy modification and low immunogenicity of aptamers make them an important alternative to traditional antibodies as biosensor recognition elements. In this book chapter, current electrochemical and optical approaches to aptamer-based biosensors are comprehensively discussed and systems based on various sensing principles are comparatively analyzed. Particular attention is paid to electrochemical, colorimetric, fluorescent, surface plasmon resonance (SPR) and lateral flow assay (LFA)-based aptasensor designs. This chapter compares different biosensor platforms in terms of sensitivity, selectivity and analytical parameters, and also includes technological advances that enable the development of new generation aptasensors. In this context, the study aims to be an up-to-date reference source for future research in the field of aptasensors.