The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) participates in viral genome packaging and abundantly produced when infected. In this work, SPR biosensor for the detection of SARS-CoV-2 in viral fluid using Fv-antibodies with the binding affinity to nucleocapsid protein (NP) of SARS-CoV-2. The F V -antibodies with a specific binding activity to the SARS-CoV-2 NP were screened using the F V -antibody library, which was expressed on the outer membrane of E. coli . F V -antibodies comprised three complementarity-determining regions (CDRs) and four frame regions (FRs) of the heavy chain at the binding pocket of IgG. The F V -antibody library was prepared by performing site-directed mutagenesis and by using the autodisplay technology; F V -antibodies with specific binding activities to the nucleocapsid protein (NP) of SARS-CoV-2 were screened using NP-immobilized magnetic beads. First, E. coli isolates with the target F V -antibody were screened, and the binding affinity (K D ) was estimated for the screened E. coli clones using FACS analysis. Then, the outer membrane (OM) of the screened E. coli clones with autodisplayed Fv-antibodies was obtained and layered on an SPR biosensor, and the binding curves of four different coronavirus (CoV) culture fluids, SARS-CoV-2, SARS-CoV, MERS-CoV, and CoV strain 229E, were compared. Finally, the F V -antibodies of the screened E. coli clones were synthesized as peptides (11 amino acid residues), and the binding constants (K D ) to NP as well as the binding curves of the CoV strains in culture fluids were estimated. Using docking simulation, binding sites and interaction types between NP and each synthetic peptide were investigated. Graphical Abstract