Objective: Currently available COVID-19 antibody tests using enzyme immunoassay (EIA) or immunochromatographic assay have variable sensitivity and specificity. Here, we developed and evaluated a novel microsphere-based antibody assay (MBA) for the detection of immunoglobulin G (IgG) against SARS-CoV-2 nucleoprotein (NP) and spike protein receptor binding domain (RBD). Method: We developed a microsphere-based assay (MBA) to determine the levels of IgG against SARS-CoV-2 NP and spike RBD. The seropositive cut-off mean fluorescent intensity (MFI) was set using a cohort of 294 anonymous serum specimens collected in 2018. The specificity was assessed using serum specimens collected from organ donors or influenza patients before 2020. Seropositive rate was determined among patients with COVID-19. Time-to-seropositivity and signal-to-cutoff (S/CO) ratio were compared between MBA and EIA. Results: MBA had a specificity of 100% (93/93; 95% confidence interval [CI], 96-100%) for anti-NP IgG and 98.9% (92/93; 95% CI 94.2-100%) for anti-RBD IgG. The MBA seropositive rate for convalescent serum specimens of COVID-19 patients were 89.8% (35/39) for anti-NP IgG and 79.5% (31/39) for anti-RBD IgG. The time-to-seropositivity was shorter with MBA than that of EIA. When compared with EIA, MBA could better differentiate between COVID-19 patients and negative controls with significantly higher S/CO ratio for COVID-19 patients and lower S/CO ratio with negative controls. MBA also had fewer specimens in the equivocal range (S/CO 0.9-1.1) than EIA. Conclusion: MBA is robust and simple, and is suitable for clinical microbiology laboratory for the accurate determination of anti-SARS-CoV-2 antibody for retrospective diagnosis, serosurveillance, and vaccine trials.