The invention discloses a preparation method of porcine small intestine antibacterial peptides PR39, which comprises the following specific steps of: 1, taking porcine small intestinal mucosa and flushing; 2, adding a mixture of acetic acid and ice water with the volume fraction of 4-5%, and smashing to be made into a homogenate; 3, carrying out a boiling water bath for 10min, and cooling to 0-10 DEG C; 4, centrifuging, cracking a supernatant by ultrasonic waves for 30s, stirring and extracting, centrifuging, and taking the supernatant; 5, mixing a precipitate with acetic acid with the volume fraction of 4-5%, stirring and extracting, centrifuging, and taking the supernatant; 6, combining the supernatants, centrifuging and taking the supernatant; 7, sampling the supernatant to a Sephadex G2100 gel column for chromatography, and collecting an eluate; and 8, detecting the antibacterial property of liquid in each collection tube by an agarose diffusion method, collecting the eluent with an antibacterial effect to obtain the porcine small intestine antibacterial peptides PR39, and storing by freezing and drying in a vacuum. The process is simple, the cost is low, most of impure proteins are removed, and the extraction rate is high.