Rutin is a potent antioxidant with therapeutic value in managing vascular and inflammatory conditions. Its accurate quantification is critical for pharmaceutical quality control and food safety. In this study, rutin was employed as a template to construct surface molecularly imprinted magnetic nanozymes (MIPs@Fe₃O₄–CoNi). These nanozymes retained peroxidase-like activity, catalyzing the H₂O₂-driven transformation of non-fluorescent terephthalic acid into fluorescent 2-hydroxyterephthalic acid. Upon rutin binding, catalytic activity was suppressed, and the fluorescence signal was further quenched through static and inner-filter effects. This dual-signal suppression mechanism significantly enhanced sensor sensitivity. The developed sensing platform exhibited a low detection limit of 0.008 μM and a broad linear range. When applied to pharmaceutical tablets, human serum, and food samples, it achieved recovery rates between 97.5 % and 104.0 %, with RSDs of 2.57 %–4.00 %. These results confirm the method's reliability, precision, and practical applicability across diverse matrices.