Surface-enhanced Raman scattering (SERS) immunoassays have emerged as highly sensitive, multiplexed analytical techniques for detecting protein biomarkers. Traditional SERS immunoassays typically rely on antibody-based SERS probes for target protein detection
however, it is challenging to obtain antibodies that are both highly effective at identifying natural proteins and suitable for SERS probe conjugation. Herein, we engineer a MultiValent Probe (MVP), consisting of multivalent nanobodies as the protein-targeting ligand to provide improved binding avidity and Raman reporter-coated gold-silver alloy nanoboxes for single-particle signal readouts. The multivalent nanobodies exhibit precise antigen recognition and exceptional affinity, and are expressed in a mammalian system for cost-effective and large-scale production. We thoroughly characterize the MVP via nanoparticle tracking analysis, nanoflow cytometry, and differential centrifugal sedimentation. To further enhance assay performance, we integrate MVP with a nanomixing-enhanced microfluidic chip to develop an MVP-based SERS-microfluidic immunoassay. As a proof of concept, we demonstrate the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins and virions from multiple strains in clinical nasopharyngeal samples (39 healthy and 39 infected), showing 84.6% concordance with RT-qPCR. This work highlights the potential of MVP-incorporated SERS-microfluidic immunoassays for diagnostics of pandemic diseases and broader applications in detecting a wide range of viral pathogens.