<p>Harmful cyanobacteria are capable of producing hepatotoxic microcystins (MCs), which pose a significant risk to both aquatic ecosystems and public health. Since cyanobacterial strains that produce MCs harbor the mcy gene cluster, monitoring and analyzing the levels of the mcy gene is critical for identifying potential MCs-producing strains and assessing periods of elevated risk. In this study, Fe single-atom nanozymes (Fe SANs) with enhanced peroxidase-like (POD) activity were synthesized, which were then used to form a Fe SANs-DNA-magnetic bead complex (Fe SANs-DNA-MB). By integrating pre-amplification via recombinase polymerase amplification (RPA), trans-cleavage by CRISPR/Cas12a, and catalytic activity by Fe SANs, a novel triple signal amplification biosensor was developed for the detection of the microcystin synthase gene E (mcyE), a critical biomarker for MCs-producing cyanobacteria. The biosensor demonstrated significantly improved analytical performance, achieving a broad dynamic rangefor mcyE detection from 0.1 pM to 20 nM (R2 = 0.99), with a low limit of detection (LOD) of 0.05 pM. Furthermore, the biosensor provides high accuracy, as evidenced by spiked recoveries of mcyE in natural lake water samples ranging from 97.93% to 103.3%. It also enabled the selective identification of the mcyE gene in toxin-producing cyanobacterial strains (FACHB-905 and FACHB-979), thereby effectively distinguishing between toxin-producing and non-toxin-producing cyanobacteria. This innovative approach advances the field of molecular biosensing by providing a highly sensitive platform for the quantitative detection of the mcyE gene, facilitating accurate monitoring of MCs-producing cyanobacteria in environmental contexts. The method holds considerable promise for enhancing water quality management and protecting public health.</p> Graphical Abstract <p></p>