• A fluorescent aptasensor based on multivalent aptamer-functionalized magnetic beads was constructed for targeted detection of P. aeruginosa . • In-situ synthesis of multivalent aptamers on magnetic beads via rolling circle amplification (RCA). • Synergistic effect of multivalent aptamers significantly enhances P. aeruginosa capture efficiency. • A simple "magnetic separation-signal-on" strategy enables convenient and highly sensitive detection of P. aeruginosa. Pseudomonas aeruginosa ( P. aeruginosa ), an important opportunistic pathogen responsible for severe infections and life-threatening diseases, is an underestimated foodborne threat to food safety and public health. Rapid and accurate monitoring in food matrices is therefore crucial. Consequently, there is a pressing need to develop highly sensitive detection methods for this pathogen. Here, we developed a fluorescent aptasensor based on multivalent aptamer-functionalized magnetic beads. The multivalent aptamers were in-situ synthesized on the surface of magnetic beads through rolling circle amplification technology. The capture ability of multivalent aptamers toward P. aeruginosa was significantly enhanced by synergistic effects. The carboxyfluorescein-labeled complementary sequences were hybridized with the multivalent aptamers to form a sensing platform. In the presence of P. aeruginosa , it competitively bound to the aptamers, triggering the dissociation of the complementary sequences. After magnetic separation, the fluorescence intensity of the supernatant increased, enabling quantitative detection of P. aeruginosa . By integrating multivalent aptamers and magnetic separation technology, the aptasensor exhibited a linear range of 10¹ – 10⁷ CFU/mL for P. aeruginosa , with a limit of detection as low as 2 CFU/mL. Furthermore, the aptasensor was successfully applied for the detection of P. aeruginosa in milk samples, with recoveries ranging from 89.33% – 106.35%. These results demonstrate that the aptasensor exhibits satisfactory analytical performance and promising practical application potential.