Unprecedented H5N1 highly pathogenic avian influenza (HPAI) outbreaks are occurring around the world and there is growing interest in the use of vaccines in affected regions. Vaccination when properly applied can contribute to HPAI control by significantly reducing virus shedding and breaking the transmission chain, but it requires robust surveillance to ensure that international trade is not affected. Thus, it is imperative to establish a test to differentiate vaccinated only animals from vaccinated and then infected animals (DIVA). In this study, we applied enzyme-linked lectin assay (ELLA) to specifically detect N1 neuraminidase (NA) antibody by inhibition of NA activity and provide a proof-of-concept bench validation using reference and experimental serum samples. We used a wild-type low pathogenic H7N1 virus of North American lineage as the ELLA antigen. The NA inhibition ELLA (NI-ELLA) was evaluated for its specificity and sensitivity using reference and experimental samples. The results demonstrated that the NI-ELLA was highly specific with low background NI activity against influenza-negative sera from different species although varying level of cross-reactivity was observed against sera of different NA subtypes with highest cross-reactivity against N4 subtype sera. Using a conservative positive cut-off threshold of 50% NI activity, NI-ELLA provides 100% specificity with all reference sera of 9 different NA subtypes. The relative sensitivity of NI-ELLA was evaluated in detecting H5N1 infection in vaccinated and then challenged birds and NI-ELLA showed higher detection rate of H5N1 infection compared with commercial NP ELISAs and real-time RT-PCR. Overall, the NI-ELLA shows high specificity and sensitivity and has the potential for application in DIVA surveillance with further validation.