The nature of biosensing is a biochemical reaction. DNA‐based chemical reaction networks (DNA‐CRNs) as a powerful programming language for describing behaviors of chemical reactions have shown great potential in designs and applications of biosensors. Due to their programmability, modularity, and versatility of the DNA strand, the performance of different detection strategies can be improved mainly by the rational design of DNA‐CRNs. Herein, an overview of the fundamental theory and biosensing processes of DNA‐CRNs is provided. Various detection strategies of DNA‐CRNs are introduced, either in a simple low‐order reaction model or in a complicated high‐order reaction type, in combination with some typical cases for the different detection purposes. In addition, an overview of the recent development of DNA‐CRNs for monitoring the cell microenvironment is presented, which is of significance to uncover some specific cell behaviors and functions. Finally, the roles of DNA‐CRNs in the rational design of high‐performance biosensors are summarized by pointing out the remaining challenges that impede the precise biosensing using DNA‐CRNs in complicated biological environments.