<p>Living bacteria can serve as biosensors for the detection of DNA in vitro and in vivo, capitalizing on their inherent ability to take up and process foreign DNA. Such bactosensors can be engineered to analyse environmental DNA, down to the single-base level, from unprocessed samples, and provide a detectable output, such as fluorescence, antibiotic resistance or therapeutic release. In this Review, we first outline design strategies for bactosensors, including genetic toolkits, such as clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems, and applications in biomedicine, agriculture, and food and water safety. Moreover, we examine chassis species, DNA uptake mechanisms, signal transduction and output strategies for bacterial biosensors intended for DNA analysis. We then consider performance metrics, including limit of detection, specificity and multiplexing, and provide a comparison between living and in vitro DNA biosensors for various applications, highlighting differences in sample processing, equipment, DNA integrity, theranostics and biocontainment.</p>