Microfluidic diagnostic devices have the potential to transform the practice of medicine. We engineered a multiplexed digital-analog microfluidic platform for the rapid and highly sensitive detection of 3-4 biomarkers in quadruplicate in 16 independent and isolated microfluidic unit cells requiring only a single 5 mu L sample. We comprehensively characterized the platform by performing single enzyme and digital immunoassays, achieving single molecule detection and measured as low as similar to 10 fM (330 fg/mL) GFP in buffer and similar to 12 fM GFP in human serum. We applied our integrated digital detection mechanisni to multiplexed detection Of 1pM anti-Ebola IgG in human serum and were able to differentiate three common Ebola strains. To ascertain that the device can be applied in environments beyond clinical point-of-care settings, we developed a low-cost, portable hardware system to control and read out the microfluidic device and detected anti-Ebola IgG in ultralow volume whole blood samples to levels of 100 pM in a multiplexed assay format.