Nipah virus (NiV), a pathogen with pandemic potential, lacks approved treatments or vaccines, highlighting the urgent need for research on immune-targeted antigenic determinants. A significant gap persists in NiV research, as studies on the fusion (F) protein critical for viral entry as well as the B cells epitopes, have primarily focused on computational prediction rather than experimental validation of immunogenic epitopes obtained through immunoinformatics approach. This study focused on the conserved F protein across NiV human isolates and employed an immunoinformatics approach to predict linear B-cell epitopes capable of direct immune activation. Predicted epitopes were screened for antigenicity, toxicity, and allergenicity. Molecular docking analysis was performed to evaluate its binding affinity with B-cell receptors (BCRs). To validate its immunogenicity, the LF6 peptide was synthesized and used in an indirect ELISA to test sera from cohort previously infected with NiV as well as with negative cohort. Epitope LF6 (LISNIEIGFCL) was identified as a strong candidate based on its immunogenic properties. Molecular docking showed favorable binding of LF6 with BCR. ELISA results revealed that one sera from the survival cohort showed positive response which is an IgG antibody response 2-fold higher (0.343) than the cut-off value (0.0171). This study provides the first reported evidence linking computational predictions with functional immune reactivity for a B-cell epitope of NiV. The findings suggest that LF6 has the potential to elicit specific immune responses. However, given the small sample size, further validation in larger cohorts is essential to confirm LF6’s vaccine relevance.