The invention relates to an O-type foot-and-mouth disease virus antibody chemiluminescence detection kit. The kit includes an polystyrene board coated by antigen, a monoclonal antibody, a standard substance, a luminescence substrate solution A and a luminescence substrate solution B. The polystyrene board coated by antigen is an opaque polystyrene plate coated by an O-type foot-and-mouth disease virus RE2 recombinant protein; the monoclonal antibody is a horseradish peroxidase labeled O-type foot-and-mouth virus monoclonal antibody; the luminescence substrate solution A comprises a Lumino, hydroxycoumarin, gallic acid, a Tris-Hcl buffer and water; the standard substance is foot-and-mouth virus O-type antibody respectively diluted buy a calibration diluent, wherein the dilution is 0NU / mL, 2NU / mL, 5NU / mL, 10NU / mL, 30NU / mL and 60NU/ mL respectively. The kit of the invention can be used for detecting O-type foot-and-mouth disease virus antibody, has the advantages of high sensitivity and wide detection range, and realizes the quantitative detection of foot-and-mouth disease antibody.