Dengue Virus (DV) is a mosquito borne disease and important public health problem in low land of Terai region in Nepal. There are limited information on the prevalence of the disease until now. This study was designed to estimate sero-prevalence of dengue virus infection in the post monsoon period (Aug-Nov) of 2008/2009 in Nepalese patients with fever visiting hospitals of Mahendranagar, Dhangadi and Dang.Serum samples were collected from 283 patients visiting hospital with history of fever & clinically suspected DF. The sero-prevalence of dengue virus specific IgM was determined by enzyme linked immunosorbent assay (ELISA) kit (PanBio, Australia).Molecular diagnosis based on RT-PCR was also performed to optimize the conditions and confirmatory diagnosis in the acute samples. The IgM capture ELISA has become the most accepted technique for the diagnosis of dengue in developing countries like Nepal.