The development of surface-enhanced Raman scattering (SERS)-based microfluidic platforms has attracted significant recent attention in the biological sciences. SERS is a highly sensitive detection modality, with microfluidic platforms providing many advantages over microscale methods, including high analytical throughput, facile automation, and reduced sample requirements. Accordingly, the integration of SERS with microfluidic platforms offers significant utility in chemical and biological experimentation. Herein, we report a fully integrated SERS-based microdroplet platform for the automatic immunoassay of specific antigen fraction 1 (F1) in Yersinia pestis. Specifically, highly efficient and rapid immunoreactions are achieved through sequential droplet generation, transport, and merging, while wash-free immunodetection is realized through droplet-splitting. Such integration affords a novel multifunctional platform capable of performing complex multistep immunoassays in nL-volume droplets. The limit of detection of the F1 antigen for Yersinia pestis using the integrated SERS-based microdroplet platform is 59.6 pg/mL, a value approximately 2 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays. This assay system has additional advantages including reduced sample consumption (less than 100 μL), rapid assay times (less than 10 min), and fully automated fluid control. We anticipate that this integrated SERS-based microdroplet device will provide new insights in the development of facile assay platforms for various hazardous materials.