Abstract: The planetary outbreak of COVID-19 has led to a substantial death toll and has hindered the functioning of modern society. This underlines the importance of accurate, cost-effective serological antibody tests and point-of-care diagnostics to monitor the viral spread and to contain pandemics and endemics. This requires a cost-effective three-dimensional nanofluidic device for rapid and multiplexed detection of viral antibodies. The device is configured to size-dependently immobilize particles at predefined positions from a multiparticle mixture, giving rise to distinct trapping lines. It is shown with the device that distinctive lines can be used as an on-chip and on-bead fluorescent linked immunosorbent assay for the detection of immunoglobulin G (IgG) antibodies against the receptor-binding domain of SARS-CoV-2 in human serum.