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The explosive spread of Zika virus (ZIKV) and associated complications in flavivirus-endemic regions underscore the need for sensitive and specific serodiagnostic tests to distinguish ZIKV, dengue virus (DENV) and other flavivirus infections. Compared with traditional envelope protein-based assays, several nonstructural protein 1 (NS1)-based assays showed improved specificity, however, none can detect and discriminate three flaviviruses in a single assay. Moreover, secondary DENV infection and ZIKV infection with previous DENV infection, both common in endemic regions, cannot be discriminated. In this study, we developed a high-throughput and multiplex IgG microsphere immunoassay (MIA) using the NS1 proteins of DENV1-DENV4, ZIKV and West Nile virus (WNV) to test samples from reverse-transcription-polymerase-chain reaction-confirmed cases, including primary DENV1, DENV2, DENV3, WNV and ZIKV infections, secondary DENV infection, and ZIKV infection with previous DENV infection. Combination of four DENV NS1 IgG MIAs revealed a sensitivity of 94.3% and specificity of 97.2% to detect DENV infection. The ZIKV and WNV NS1 IgG MIAs had a sensitivity/specificity of 100%/87.9% and 86.1%/78.4%, respectively. A positive correlation was found between the readouts of enzyme-linked immunosorbent assay and MIA for different NS1 tested. Based on the ratio of relative median fluorescence intensity of ZIKV NS1 to DENV1 NS1, the IgG MIA can distinguish ZIKV infection with previous DENV infection and secondary DENV infection with a sensitivity of 88.9–90.0% and specificity of 91.7–100.0%. The multiplex and high-throughput assay could be applied to serodiagnosis and serosurveillance of DENV, ZIKV and WNV infections in endemic regions., Author summary Although there was a decrease of Zika virus (ZIKV) infection since late 2017, the specter of congenital Zika syndrome and its re-emergence in flavivirus-endemic regions emphasize the need for sensitive and specific serological tests to distinguish ZIKV, dengue virus (DENV) and other flaviviruses. Compared with traditional tests based on envelope protein, several nonstructural protein 1 (NS1)-based assays had improved specificity, however, none can discriminate three flaviviruses in a single assay. Moreover, secondary DENV infection and ZIKV infection with previous DENV infection, both common in endemic regions, cannot be distinguished. Herein we developed a high-throughput and multiplex IgG microsphere immunoassay using the NS1 proteins of four DENV serotypes, ZIKV and West Nile virus to test samples from laboratory-confirmed cases with different primary and secondary flavivirus infections. Combination of four DENV NS1 assays revealed a sensitivity of 94.3% and specificity of 97.2%. The ZIKV and WNV NS1 assays had a sensitivity/specificity of 100%/87.9% and 86.1%/78.4%, respectively. Based on the signal ratio of ZIKV NS1 to DENV1 NS1, the assay can distinguish ZIKV infection with previous DENV infection and secondary DENV infection with a sensitivity of 88.9–90.0% and specificity of 91.7–100.0%. This has applications to serodiagnosis and serosurveillance in endemic regions.