Exosomes are cell-derived nano-sized vesicles that have been recently recognized as new mediators for many cellular processes and potential biomarkers for non-invasive disease diagnosis and the monitoring of treatment response. To better elucidate the biology and clinical value of exosomes, there is a pressing need for new analytical technologies capable of the efficient isolation and sensitive analysis of such small and molecularly diverse vesicles. Herein, we developed a microfluidic exosome analysis platform based on a new graphene oxide/polydopamine (GO/PDA) nano-interface. To the best of our best knowledge, we report for the first time, the GO-induced formation of a 3D nanoporous PDA surface coating enabled by the microfluidic layer-by-layer deposition of GO and PDA. It was demonstrated that this nanostructured GO/PDA interface greatly improves the efficiency of exosome immuno-capture, while at the same time effectively suppressing non-specific exosome adsorption. Based on this nano-interface, an ultrasensitive exosome ELISA assay was developed to afford a very low detection limit of 50 μL−1 with a 4 log dynamic range, which is substantially better than the existing methods. As a proof of concept for clinical applications, we adapted this platform to discriminate ovarian cancer patients from healthy controls by the quantitative detection of exosomes directly from 2 μL plasma without sample processing. Thus, this platform could provide a useful tool to facilitate basic and clinical investigations of exosomes for non-invasive disease diagnosis and to aid precision treatment.