Antibiotic and heavy metal contamination in water poses a severe global public health risk, yet conventional detection methods remain constrained by reliance on complex laboratory instrumentation. Although cell-free biosensors utilizing allosteric transcription factors (aTFs) offer promising alternatives, their limited sensitivity impedes reliable detection of small-molecule contaminants. To address this limitation, we developed allosteric transcription factors (aTFs)-regulated rolling circle transcription assay (ARCTA) for ultrasensitive detection of water contaminants. The ARCTA system employs a circular DNA template comprising a double-stranded region with a T7 promoter and aTFs binding sequence, alongside a single-stranded domain transcribable into fluorescent RNA. Target-specific recognition by aTFs enables rapid, efficient transcription and amplification of fluorescent RNA, achieving an exceptionally low detection threshold. Applied to tetracycline, oxytetracycline, erythromycin, clarithromycin, Hg