The invention provides an extraction method of biological membrane proteins and a preparation method of an electrophoresis sample. The extraction method comprises the following steps: pretreating a biological membrane sample to obtain a precipitate; mixing the precipitate with a pyrolysis solution and a phenylmethylsulfonyl fluoride solution, and performing ultrasonic pyrolysis under an ice bath condition to obtain a sample pyrolysis solution; and shaking and centrifuging the sample pyrolysis solution, taking a supernatant, and treating the supernatant by adopting a TCA-acetone precipitation method, thereby obtaining a protein sample. The preparation method comprises the following steps: dissolving the protein sample by using a dissolving solution, and sequentially performing ultrasonic treatment, vortex oscillation and centrifugation, and taking a supernatant to obtain the electrophoresis sample. According to the methods provided by the invention, the pyrolysis solution containing various detergents is adopted and is combined with the ice bath ultrasound to break walls of microbial cells, and meanwhile, the TCA-acetone precipitation method is adopted to purify the protein, so that whole proteins in the biological membrane sample can be effectively extracted, and the masking action of humus to the proteins can be reduced to ensure that the analysis result of electrophoresis is clear.