<p>Onsite and sensitive detection of microcystin-LR (MC-LR) is crucial for monitoring environmental pollution and protecting human health, particularly in the context of increasing cyanobacterial harmful algal blooms (CyanoHABs). Herein, this study reports a rolling circle transcription (RCT)-based fluorescent aptasensor designed for rapid and quantitative onsite detection of MC-LR in real water samples. Aptamers specific to MC-LR were immobilized onto magnetic beads (MBs) and hybridized with blocker complementary DNA strands to form the MB aptasensor complex. Upon recognition and binding of MC-LR molecules in water samples, the blocker DNA strands were displaced and subsequently cyclized into circular DNA structures via T4 DNA ligase-mediated ligation. These circular DNA templates initiated RCT, generating abundant fluorescent Mango RNA reporters. The resulting fluorescent signals exhibited a robust positive correlation with MC-LR concentration, with a detection limit of 39 pM and a linear range from 0.01 to 3000 nM, demonstrating high sensitivity and specificity. The aptasensor showed excellent specificity against structural analogs and common interferents. Recovery tests in Haihe River samples yielded rates between 98.33% and 101.17%, with relative standard deviations below 5%. This sensing platform offers a simple operational procedure, minimal sample preparation, and excellent applicability for real environmental water monitoring. Thus, the developed RCT-based aptasensor provides a promising, cost-effective alternative for onsite MC-LR detection and contributes significantly to environmental safety and public health management.</p> Graphical abstract <p></p>