Flow cytometry has emerged as a promising technique for detection of SARS-CoV-2 antibodies. In this study, we described a new methodology to detect simultaneously IgG, IgM and IgA of SARS-CoV-2 nucleocapsid protein in human serum by flow cytometry. The Nucleocapsid protein was covalently bound on functional beads surface applying sulfo-SMCC chemistry. BUV395 anti-IgG, BB515 anti-IgM, biotinylated anti-IgA1/IgA2 and BV421 streptavidin were used as fluorophore conjugated secondary antibodies. Serum and antibodies reaction conditions were optimized for each antibody isotype detection and a multiplexed detection assay was developed. This new cell-free multiplex approach based on flow cytometry was able to efficiently discriminate COVID-19 negative and positive samples. The simultaneous detection of IgG, IgM and IgA showed a sensibility of 88.5-96.2% and specificity of 100%. The combined detection of antibody isotypes offers greater spectrum for detection and monitoring of COVID-19 vaccines and seroconversion. This novel strategy opens a new avenue for flow cytometry-based diagnosis.