The invention discloses an O type foot-and-mouth disease 146S antigen quantitative ELISA (enzyme-linked immuno sorbent assay) detection kit and a method for using the same. The kit comprises an ELISA plate, an O type foot-and-mouth disease standard reference antigen, a demulsifier, an O type foot-and-mouth disease rabbit antiserum, an O type foot-and-mouth disease guinea pig antiserum, a rabbit anti-guinea pig-horse radish peroxidase conjugate, a guinea pig antiserum dilute solution, a 25-fold PBST (phosphate buffer solution tween) concentrated solution, a carbonate buffer solution capsule, a citric acid-phosphate buffer solution tablet, an OPD (o-phenylenediamine) tablet, a stop solution, a plate sealing membrane, a moving liquid tank and a 96-mesh U-shaped dilution plate. The kit is an organic combination of a sucrose density gradient centrifugation method and an indirect sandwich ELISA method, integrates the advantages of the sucrose density gradient centrifugation method and the indirect sandwich ELISA method, is simple to operate and good in stability, is suitable for batch detection, can be used for distinguishing serum types, and is an ideal substitution method for antigen quantitative and vaccine efficacy detection.