Although exhibiting no or low virulence in poultry, avian influenza virus H7N9 has caused around 1,200 confirmed human infections in China with a case-fatality rate of 30% since 2013. A highly pathogenic H7N9 virus (HP-H7), with the HA antigenicity distinct from the previous, were recently detected in patients and poultry. Therefore, convenient rapid diagnosis with reliability will allow early antiviral use and management for H7N9 infection. Here, a sandwich ELISA targeting the conserved viral antigen, neuraminidase (NA) was developed. The immunoassay employed mouse monoclonal antibody (mAb) 3C1 to specifically capture the N9 and 3E9 for the detection. Its limit of detection is 6.25ng/ml for N9 protein of A/Anhui/1/2013(H7N9, AH1/2013) and 0.125HAU/50μl for live virus, AH1/2013 and A/Environment/Jiangxi/28/2009 (H11N9), respectively. When applied to test the five clinic throat swabs from H7N9 patients confirmed by nuclear acid testing (NAT) using quantitative reverse-transcriptase polymerase chain reaction (Q-PCR), two samples showed positive result in sandwich ELISA while all were negative using commercial Flu A and H7 subtype rapid antigen tests (RAT). The ELISA using anti-N9 mAbs provided a valuable approach to detect H7N9 virus and quantify the N9 protein.