Performing bioassay formats based on enzyme and antibody recognition reactions with a single detection chip remains an unmet challenge owing to the different requirements of such bioassays. Herein, we describe a dual-marker biosensor chip, integrating enzyme and antibody-based assays for simultaneous electrochemical measurements of insulin (I) and glucose (G). Simultaneous G/I sensing has been realized by addressing key fabrication and operational challenges associated with the different assay requirements and surface chemistry. The I immunosensor relies on a peroxidase-labeled sandwich immunoassay, while G is monitored through reaction with glucose oxidase. The dual diabetes biomarker chip offers selective and reproducible detection of picomolar I and millimolar G concentrations in a single microliter sample droplet within less than 30min, including direct measurements in whole blood and saliva samples. The resulting integrated enzymatic-immunoassay biosensor chip opens a new realm in point-of-care multiplexed biomarker detection.