<p>Enzymatic assays represent a promising alternative to conventional multiplex optical methods for multi-target detection due to their exceptional specificity and catalytic versatility. Nevertheless, most existing enzymatic systems are limited to single-target detection. In this work, we developed a dual-signal multiplex assay based on non-tandem enzymes for the simultaneous detection of multiple pathogenic bacteria. This assay utilizes the distinct catalytic activities of horseradish peroxidase (HRP) and invertase (INV) to produce two orthogonal signal outputs. Specifically, HRP catalyzes the oxidation of 3,3′,5,5′-tetramethylbenzidine to produce a blue chromogenic signal measurable by absorbance, while INV hydrolyzes sucrose to yield glucose, which is quantified with a personal glucose meter. To implement this assay, HRP and INV are individually encapsulated within zeolitic imidazolate framework-90, and Fe₃O₄ nanoparticles functionalized with phenylboronic acid are employed for dual-recognition of target pathogens. By integrating spatiotemporal control of enzymatic reactions with distinct signal separation, the assay effectively minimizes crosstalk and enhances detection accuracy and reliability. This assay demonstrates excellent sensitivity and selectivity in milk samples, highlighting its potential for practical applications. We believe this work establishes a versatile and robust platform for multiplex pathogen detection, with significant implications for food safety, clinical diagnostics, and environmental monitoring.</p> Graphical Abstract <p></p>