The invention discloses a hybridoma cell line secreting a foot-and-mouth disease virus non-structural protein monoclonal antibody 3A10, and an application thereof. The hybridoma cell line stably secreting the foot-and-mouth disease virus non-structural protein monoclonal antibody, with the microbial preservation number of CGMCC NO.16213, is finally obtained through screening using a cell fusion technology by immunizing mice with the foot-and-mouth disease non-structural protein. The monoclonal antibody secreted by the hybridoma cell line has the advantages of good broad spectrum, high conservatism, strong affinity and strong competitiveness with serum, and can be used as a detection antibody in a blocking ELISA detection method to accurately distinguish naturally infected animals with thefoot-and-mouth disease and immunized animals. The invention further provides a blocking ELISA detection kit established by using the monoclonal antibody as a detection antibody. The detection kit canbe adopted to detect the serum of any animal species, spans the inter-species barrier, and has the advantages of high reliability, wide application range, good broad spectrum, strong competitiveness,and quickness and convenience in detection.