There are an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotide-based biosensors are those where the probe to capture the analyte is a strand of DNA, RNA or a synthetic analogue to naturally occurring nucleic acids. This chapter will draw light upon various types of nucleic acids such as DNA, RNA (particularly microRNAs), their role and their application in biosensing. Also, it will cover DNA/RNA aptamers, which can be used as bioreceptors to a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides like PNA or LNA has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the lab-based status and the reality of biomedical applications.