Rapid, sensitive analysis of protein biomarkers is of tremendous biological and clinical significance. Immunoassays are workhorse tools for protein analysis and have been under continuous investigation to develop new methods and to improve the analytical performance. Herein we report a pneumatically gated microfluidic communicating vessel (μCOVE) chip for rapid and sensitive immunomagnetic ELISA. A distinct feature of our device is that it employs the communicating vessel principle as a simple means to generate a fast transient hydrodynamic flow to enable effective flow washing without the need for excessive incubation, which greatly simplifies and expedites the assay workflow, compared to conventional microfluidic flow-based immunoassays. Stationary multi-phase microfluidic techniques have been developed for fast bead washing. However, they have some limitations, such as the need for careful control of interfacial properties, large bead quantity required for reliable interphase bead transport, and relatively high bead loss during surface tension-gated traverse. Our single-phase μCOVE chip can overcome such limitations and facilitate the manipulation of magnetic beads to streamline the assay workflow. We showed that the μCOVE device affords highly sensitive quantification of the CEA and EGFR proteins with a LOD down to the sub-picogram per mL level. Direct detection of the EGFR in the crude A431 cell lysate was also demonstrated to further validate the ability of our device for rapid and quantitative analysis of complex biological samples. Overall, our work presents a unique platform that combines the merits of the stationary multi-phase systems and the flow-based microfluidics. This novel immunoassay microsystem has promising potential for a broad range of biological and clinical applications, owing to its simplicity and high performance.