The invention discloses a foot-and-mouth disease virus (FMDV) 2C3ABC recombinant protein as well as a preparation method and application thereof, and belongs to the field of pharmaceutical biotechnology. According to the invention, the mutation of the following sites is performed on the basis of an original FMDV 3C protease gene: Cys142-Ser, Cys163-Gly. FMDV recombinant protein 2C3ABC is expressed as an inclusion body in the bacterial cytoplasm, and is subjected to separation, denaturation, renaturation and multi-step purification, to obtain a complete and enzymolysis-free FMDV nonstructural protein mu2C3ABC, wherein the protein has solubility and complete antigenicity, and has a molecular weight of 72 kDa. The protein, as a diagnostic antigen, is prepared into chromatographic strips, has sensitivities of 98.4% and 100% respectively in the detection of FMDV experimentally infected pigs and naturally infection-free pigs, and has specificities of 100% and 98% respectively in the detection of naturally infection-free pigs and vaccine-immunized pigs. Compared with commercial kits Ceditest and UBI, the FMDV 2C3ABC recombinant protein has a high degree of consistency, can be used to distinguish infected animals and immunized animals, wherein K = 0.823 (p is smaller than 0.05).