Rapid molecular biosensing is an emerging application area for synthetic biology. Here, we engineer a portable biosensor for cyanuric acid (CYA), an analyte of interest for human and environmental health, using a LysR-type transcription regulator (LTTR) from Pseudomonas within the context of Escherichia coli gene expression machinery. To overcome cross-host portability challenges of LTTRs, we rationally engineered hybrid Pseudomonas-E. coli promoters by integrating DNA elements required for transcriptional activity and ligand-dependent regulation from both hosts, which enabled E. coli to function as a whole-cell biosensor for CYA. To alleviate challenges of whole-cell biosensing, we adapted these promoter designs to function within a freeze-dried E. coli cell-free system to sense CYA. This portable, on-demand system robustly detects CYA within an hour from laboratory and real-world samples and works with both fluorescent and colorimetric reporters. This work elucidates general principles to facilitate the engineering of a wider array of LTTR-based environmental sensors.