The invention discloses a preparation method of a recombinant antimicrobial peptide extracted from a small intestine of a pig and an application thereof. The preparation method comprises the following steps: mashing, homogenizing and repeatedly freezing and thawing the small intestine tissue of the pig with a meat grinder and a colloid mill, and preliminarily crushing cells; then performing the water boiling heat treatment, and extracting with acetic acid solution to obtain a large amount of antimicrobial peptides; centrifuging to enable the quantity of other proteins to be reduced greatly, filtering the supernate obtained through centrifuging by a tangential flow micro-filtration membrane and a tangential flow ultrafiltration membrane sequentially, adjusting the pH value, filtering the supernate by a tangential flow nanofiltration membrane, concentrating and dialyzing to desalt, and disinfecting after adjusting the osmotic pressure to obtain the recombinant antimicrobial peptide extracted from the small intestine of the pig. According to the invention, the application of the recombinant antimicrobial peptide adopts effects of the antimicrobial peptide on aspects of mucosal immunity, immunopotentiators, novel antibiotics and the like, and the mucosal immunity character of the antimicrobial peptide can play an important role in the prevention and control of transmissible gastroenteristis, epidemic diarrhea and mycoplasma gallisepticum; the antimicrobial peptide replaces traditional antibiotics, and is remarkable in effect.