The invention discloses a fusion gene of mycoplasma hyopneumoniae and a preparation method of the fusion gene, and belongs to the field of genetic engineering technology. P46 and P65 genes adopted in the invention can directly design specific primers containing corresponding restriction sites according to a coding region sequence released in NCBI GeneBank. A PCR product of the P46 gene is subject to double enzyme digestion and connection with a prokaryotic expression vector pET-28a, so as to construct a recombinant expression vector pET-28a-P46; and the recombinant expression vector pET-28a-P46 is subject to double enzyme digestion and connection with a PCR product of the P65 gene, so as to directly construct a recombinant expression vector pET-28a-P46-P65. The fusion gene and the preparation method have the following advantages: instead of Linker connection, the P46 and P65 genes are directly subject to double enzyme digestion connection so as to prepare the recombinant expression vector pET-28a-P46-P65; and compared with a process of connecting genes by virtue of Linker and then connecting to a prokaryotic expression vector, the recombinant vector construction process is simple and is easy to prepare the recombinant expression vector, and consumed time is significantly shortened.