COVID-19 pandemic was caused by the severe acute respiratory syndrome coronavirus 2 (Sars-CoV-2). The nucleocapsid (N) protein from Sars-CoV-2 is a highly immunogenic antigen and responsible for genome packing. Serological assays are important tools to detect previous exposure to SARS-CoV-2, complement epidemiological studies, vaccine evaluation and also in COVID-19 surveillance. SARS-CoV-2 N (r2N) protein was produced in Escherichia coli, characterized, and the immunological performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and beads-based array immunoassay. r2N protein oligomers were evidenced when it is associated to nucleic acid. Benzonase treatment reduced host nucleic acid associated to r2N protein, but crosslinking assay still demonstrates the presence of higher-order oligomers. Nevertheless, after RNase treatment the higher-order oligomers reduced, and dimer form increased, suggesting RNA contributes to the oligomer formation. Structural analysis revealed nucleic acid did not interfere with the thermal stability of the recombinant protein. Interestingly, nucleic acid was able to prevent r2N protein aggregation even with increasing temperature while the protein benzonase treated begin aggregation process above 55 °C. In immunological characterization, ELISA performed with 233 serum samples presented a sensitivity of 97.44% (95% Confidence Interval, CI, 91.04%, 99.69%) and a specificity of 98.71% (95% CI, 95.42%, 99.84%) while beads-based array immunoassay carried out with 217 samples showed 100% sensitivity and 98.6% specificity. The results exhibited an excellent immunological performance of r2N protein in serologic assays showing that, even in presence of nucleic acid, it can be used as a component of an immunoassay for the sensitive and specific detection of SARS-CoV-2 antibodies.