The invention belongs to the field of biotechnology and particularly relates to pichia pastoris expressed recombinant chicken Cathelicidin antibacterial peptide and further discloses a preparation method and application thereof. In the method for preparing recombinant chicken Cathelicidin antibacterial peptide disclosed by the invention, a pPICZalphaA secreted expression vector is selected as a eukaryotic expression system of the exogenous gene so that the recombinant chicken Cathelicidin antibacterial peptide is efficiently expressed and direct killing of host cells caused by the expression product is avoided. Meanwhile, the method has the advantages of high expression efficiency, simple separation and purification of expression products, low production cost, easiness in magnification and good stability and is suitable for large-scale industrial production. According to experimental verification, the combination of Cathelicidin antibacterial peptide and antibiotic realizes an obvious synergistic antibacterial effect on multiple kinds of gram-positive bacteria and gram-negative bacteria, and the application and promotion prospects are broad.