Abstract: The creatine kinase plays an important role in bovine energy metabolism. When inflammation occurs, there will be a locally-specific increase of CK. According, the creatine kinase may serve as a potential marker of bovine diseases. In this study, a total of 54 mutually overlapped primers were designed and synthesized according to the published amino acid sequence of bovine creatime kinase isoenzyme CK-BB and the codon usage preferred by E. coli（BL21）. A total of 6 DNA fragments of CK-BB gene,including CK-BB1, CK-BB2, CK-BB3, CK-BB4, CK-BB5 and CK-BB6, were synthesized by assembled PCR assay. Using the mixture of the 6 fragments as template, the second round PCR was conducted with head primer ck-1 and tail primer ck-54, and the amplifying product was connected with vector p ET28 b. The positive recombinants were subsequently identified, cloned and sequenced. A correct synthetic CK-BB gene was obtained by proofreading PCR assay, which lays a foundation for large-scale preparation of bovine isoenzyme CK-BB.