An automatic electrokinetically-controlled heterogeneous immunoassay with multiple analyte detection capability was developed in this study. Numerical simulation with the experimentally-measured mobility values was employed to study the transport processes in a microfluidic network (mFN). The operation parameters obtained from the numerical simulation was then applied to design and control the immunoassay chip. The effectiveness of the automatic electrokinetic control was proved. The immunoassay chip was made of poly(dimethylsiloxane)(PDMS)/PDMS-coated glass using photolithography and replica molding. Multiple antigen immobilization was accomplished by adsorbing the antigen molecules onto the PDMS film coated on a glass slide and by using a mFN. The performance of the immunoassay was assessed using several microbial antigens. Escherichia coli O157: H7 lysate antigen could be reliably detected in a concentration range of 3~20 mg/mL. The assay also displayed very good specificity, when the primary and secondary antibodies used were mixtures of different species. The time for the immunoassay, from antigen coating to signal detection, was only one hour. While still an un-optimized prototype, this automatic, high-throughput immunoassay chip shows a great potential in detecting multiple pathogenic infections efficiently for clinical applications.