<p>A microfluidic analytical system based on magnetically retained nanomaterials has been developed using fluorometric detection. This system utilizes magnetic gold nanoclusters (AuMNCs) to determine total tetracyclines using three of them as model—chlortetracycline (CTC), doxycycline (DC), and oxytetracycline (OTC)—in water samples by monitoring the change in the luminescent signal produced upon interaction. AuMNCs were synthesized by adding preformed magnetic nanoparticles (MNPs) to bovine serum albumin (BSA) as stabilizer and ascorbic acid as a reducing agent. Their magnetic properties enable their retention in the reaction/detection zone of the microfluidic system. The reactor was integrated into the optical pathway of a conventional spectrofluorometer with a 3D-printed device to align the excitation beam with the microchannel. The instrumental signal was obtained at a 22.5° angle from the excitation beam. Key parameters were studied, including pH (optimal at 11 with carbonate buffer), flow rate (40 µL·min⁻¹), and AuMNCs injection volume (10 µL). The analytical signal was based on the fluorescence intensity ratio between the emission of the AuMNCs (650&#xa0;nm) and the AuMNCs-tetracycline complexes (425 or 510&#xa0;nm). Limits of detection of 0.41&#xa0;µmol·L⁻¹ for CTC, 0.70&#xa0;µmol·L⁻¹ for DC, and 0.61&#xa0;µmol·L⁻¹ for OTC, with relative standard deviations (RSD) below 6.2%, were achieved. Compared with non-retained AuMNCs, the magnetically retained system showed improved results. The system was applied to determine different tetracyclines in water samples, with recoveries in spiked river water samples from 86 to 111%. This approach offers a simple, label-free, and versatile alternative with potential for future development into multi-analyte sensing platforms or applications in complex matrices.</p> Graphical Abstract <p></p>