Synthetic peptides have recently become common as antigens for antibody production. Peptides are short chains of amino acids that can be used to elicit an immune response. The immunogenicity of the peptide antigens varies depending on the length, charge, solubility, and amino acids contained in the peptide sequence. Dengue NS1 protein is an important target antigen in the early detection of dengue infection. In this study, peptides corresponding to a highly conserved region from the dengue NS1 region were designed and synthesized. Balb/C mice were immunized against each peptide and spleen cells extracted from the immunized mice were fused with NS0 murine myeloma cells. Hybridoma clones obtained from the fusions were tested against peptides using ELISA. Out of 1,830 growing clones, 28 clones produced antibodies reacting with dengue NS1 peptides. A purified monoclonal antibody reacting with all four peptides was tested for reactivity with dengue NS1 native protein using dengue-confirmed serum and urine samples. The monoclonal antibody shows significant reactivity with both serum and urine. The findings of the current research can be used to detect dengue infection using urine, which ultimately results in the prevention of dengue epidemics through painless diagnosis, following treatment, and patient management to safeguard human and economic wellness.