The invention discloses Mycoplasma hyopneumoniae P97R1 gene recombined Pichia pastoris and expression protein, and relates to the field of bioprotein preparation. The invention is mainly obtained by gene acquisition, establishment of an expression vector and expression of target protein. A method for preparing the protein comprises the following steps that: a PCR method and uses a designed primer to obtain the target protein from Mycoplasma hyopneumoniae genome DNA by amplification; the target gene is cloned into a yeast expression vector pPICZalpha-A through enzyme restriction and connection; a secretion type recombined yeast expression vector pPICZalpha-A/P97R1 is established; and the pPICZalpha-A/P97R1 is electrically converted into a Pichia pastoris strain GS115 through Sac I enzyme restriction and linearization. The recombined Pichia pastoris expresses the P97R1 protein through methanol induction and secretion. The Mycoplasma hyopneumoniae P97R1 protein prepared by the method is pure, has good immunoreaction, and can be used for researching Mycoplasma hyopneumoniae P97 protein and developing Mycoplasma hyopneumoniae immunodetection kits and genetic engineering vaccines.