The indirect ELISA is a simple and useful method for detection of pathogen-specific antibodies in animal sera. However, non-specific or background binding is often a problem, especially when recombinant proteins from Escherichia coli are used. In this study, a comparative indirect ELISA in which the total reactivity and the background binding were determined simultaneously on the same ELISA plate was reported. The background was determined by incubation of the test sera with excess free antigen to block specific binding. The sample was considered positive only when its total reactivity reading was higher than a pre-determined cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Using this approach, an antibody assay for henipaviruses using a recombinant Nipah virus nucleocapsid protein expressed in E. coli was developed. A total of 919 negative serum samples were tested in this assay and the specificity was 95.8%. In addition, eight positive experimental serum samples all tested positive. The use of recombinant protein as the ELISA antigen, instead of inactivated virus antigens, will be of significant advantage for countries where there is no facility of Biosafety level 4 to handle this group of zoonotic viruses.