The dengue virus (DV) causes one of the most important arthropod-borne human viral diseases throughout the tropical and subtropical countries. However, the morbidity and mortality of DV infections could be reduced with an early hospitalization care and a rapid risk identification of developing the dengue haemorrhagic fever (DHF). The nonstructural glycoprotein 1 (NS1) has been pointed as a reagent for immune-assay diagnostic test optimization. To evaluate this potential, recombinant DV2-NS1 proteins (rNS1) were produced from Escherichia coli (NS1EC) and insect cells (NS1IC) expression. The tests were performed by analysis of a human serum panel reacted against different rNS1 forms. The results demonstrated high correspondence between the DV positive sera and the assay results using native or refolded forms of either NS1IC or NS1EC. Also, the IgG and IgM anti-rNS1 level profiles showed distinct distribution, depending on protein form and disease status. However, the IgM anti-rNS1 reactions did not show sensibility to detect the DV in primary infections. The data obtained from the paired serum samples reactivity comparison suggested a heterogeneous human immune response and absence of correspondence between the IgG and IgM profile levels. Moreover, a patient with negative reference test could be detected by specific IgG anti-rNS1 assays presented here. Therefore, these results sustain the usefulness of dengue nonstructural proteins, in particular the NS1, in diagnostic tests as a complementary reagent.