The purpose of this study was to characterize the diagnostic performance of a newly developed enzyme-linked immunosorbent assay (ELISA) for detection of SARS-CoV-2 nucleocapsid protein (NP) in blood. Blood samples were collected during hospitalization of 165 inpatients with PCR-confirmed SARS-CoV-2 infection and from 505 outpatients predominantly with relevant symptoms of COVID-19 simultaneously with PCR testing. For the 143 inpatients who had their first blood sample collected within 2 weeks after PCR-confirmed infection, the diagnostic sensitivity of the ELISA was 91.6%. The mean NP concentration of the 131 ELISA-positive blood samples was 1,734 pg/ml (range, 10 to 3,840 pg/ml). An exponential decline in NP concentration was observed for 368 blood samples collected over the first 4 weeks after PCR-confirmed SARS-CoV-2 infection, and all blood samples taken later had an NP concentration below the 10-pg/ml diagnostic cutoff. The diagnostic sensitivity of the ELISA was 81.4% for the 43 blood samples collected from outpatients with a simultaneous positive PCR test, and the mean NP concentration of the 35 ELISA-positive samples was 157 pg/ml (range, 10 to 1,377 pg/ml). For the 462 outpatients with a simultaneous negative PCR test, the diagnostic specificity of the ELISA was 99.8%. In conclusion, the SARS-CoV-2 NP ELISA is a suitable laboratory diagnostic test for COVID-19, particularly for hospitals, where blood samples are readily available and screening of serum or plasma by ELISA can facilitate prevention of nosocomial infections and reduce the requirement for laborious swab sampling and subsequent PCR analysis to confirmatory tests only.