The RNA Output Sensors Activated by Ligand Induction (ROSALIND) system consists of three programmable components: highly-processive RNA polymerases, allosteric transcription factors and synthetic DNA transcription templates. Together, these components allosterically regulate the in vitro transcription of a fluorescence-activating RNA aptamer: in the absence of an analyte of interest, transcription is blocked, while in its presence, a fluorescent signal is produced. This design is compatible with downstream RNA circuitry that can tune the specificity and sensitivity of transcription factor outputs. ROSALIND can be freeze-dried for field deployment, thereby creating water quality sensors that are activated upon rehydration with the sample of interest. A low-cost, 3D-printed handheld device provides easy visualization of the sensor’s RNA output.