The availability of random peptide libraries displayed on bacteriophage (RPL) has provided a powerful tool for selecting sequences that mimic binding properties of natural antigen epitopes (mimotopes). These mimotopes can be used for vaccine design, drug development, and diagnostic assays. Several mimotopes have been shown to induce production of antibodies against the natural antigen. We have previously identified four dengue virus mimotopes from a phage-displayed peptide library using antidengue 3 human sera. Three of them showed similarity in their amino acid sequences with the NS4b proteins of dengue. Few studies have examined the role of NS4b proteins in the antibody response to dengue virus infection. A multiple antigen peptide (MAP) system was chemically synthesized containing this mimotope (NS4b MAP), and BALB/c mice were immunized to evaluate its immunogenicity. Antipeptide responses were induced and recognised DENV-3 infected cells as determined by immunofluorescence. The high levels of the IgG2a subtype against NS4bMAP suggest the induction of a Th1-like response. Our findings suggest that the NS4b mimotope might be a useful tool for the development of multiepitope diagnostic assays, dengue virus vaccine design, and pathogenesis studies.