In this study, a multifunctional aptamer-conjugated magnetic covalent organic framework (COF)-CuO/Au nanozyme (MCOF-CuO/Au@apt) was developed as a “three-in-one” platform for dual-signal colorimetric and fluorescent detection of Vibrio parahaemolyticus. The nanozyme integrated magnetic separation, peroxidase-like catalytic activity, and specific target recognition through an aptamer-based strategy. Upon binding to V. parahaemolyticus, the catalytic oxidation of tetra-aminophenylethylene (TPE-4A) by the nanozyme was selectively inhibited, resulting in distinct colorimetric and fluorescent signals that significantly enhanced the detection accuracy and reliability. The proposed method exhibited high sensitivity, with limits of detection (LOD) of 21 and 7 CFU/mL for the colorimetric and fluorescent assays, respectively. The performance of this method was validated using real seafood samples, including Penaeus vannamei, Mytilus coruscus, and Crassostrea gigas, which showed high recovery rates (101.11%–107.30%) and excellent reproducibility. The system also demonstrated strong specificity and accuracy under various conditions, confirming its robustness and practical applicability. Collectively, this innovative platform presents a promising solution for the rapid, versatile, and sensitive detection of V. parahaemolyticus in seafood, with considerable potential to advance food safety diagnosis and on-site monitoring.