As the COVID-19 pandemic escalates, the need for effective vaccination programs, therapeutic intervention, and diagnostic tools increases. Here, we identified 11 unique nanobodies (Nbs) specific for the SARS-CoV-2 spike receptor-binding domain (RBD) of which 8 Nbs potently inhibit the interaction of RBD with angiotensin-converting enzyme 2 (ACE2) as the major viral docking site. Following a detailed epitope determination and characterization of the binding mode by structural analysis, we constructed a hetero-bivalent Nb targeting two different epitopes within the RBD:ACE2 interface. This resulted in a high-affinity binder with a viral neutralization efficacy in the picomolar range. Using the bivalent Nb as a surrogate, we established a competitive multiplex binding assay (“NeutrobodyPlex”) for detailed analysis of the presence and performance of neutralizing RBD-binding antibodies in the serum of convalescent or vaccinated patients. As demonstrated, the NeutrobodyPlex enables high-throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, helping to monitor immune status or guide vaccine design. This approach is easily transferrable to diagnostic laboratories worldwide.