Aptamers are short oligonucleotides isolated in vitro from randomized libraries that can bind to specific molecules with high affinity, and offer a number of advantages relative to antibodies as biorecognition elements in biosensors. However, it remains difficult and labor‐intensive to develop aptamer‐based sensors for small‐molecule detection. Here, we review the challenges and advances in the isolation and characterization of small‐molecule‐binding DNA aptamers and their use in sensors. First, we discuss in vitro methodologies for the isolation of aptamers, and provide guidance on selecting the appropriate strategy for generating aptamers with optimal binding properties for a given application. We next examine techniques for characterizing aptamer–target binding and structure. Afterwards, we discuss various small‐molecule sensing platforms based on original or engineered aptamers, and their detection applications. Finally, we conclude with a general workflow to develop aptamer‐based small‐molecule sensors for real‐world applications. Aptamers, oligonucleotide‐based recognition elements isolated from random libraries, have several favorable attributes for biosensing. This Review examines state‐of‐the‐art methods and advances in the isolation and characterization of small‐molecule‐binding aptamers and their use in various biosensors. Factors limiting aptamer‐based sensors and potential solutions to these issues are also discussed.