The invention relates to a preparation method of a biological preservative. As known to all, along with the rapid development of the society, chemical preservatives have been used in food, have strong impact on food safety and endanger the health of consumers, therefore, the use of preservatives causes great concern of government departments and consumers. According to the biological preservative, natural plants and microbial antibiotic substances are used as base materials, for example, the biological preservative is prepared by mixture of carboxymethyl chitosan, xylooligosaccharide, nisin from lactic streptococci, acetic acid, lactobacillus reuteri, pediococcus acidilactici, calcium citrate, ionized calcium, lactic acid, plant essential oil and water. The biological preservative can inhibit growth of harmful microorganisms by antagonistic and competitive inhibition effects among different microorganisms and produce extracted and separated components of antibacterial active ingredients having antifungal and antibacterial functions in normal growth and metabolism processes, thereby achieving antiseptic and fresh-keeping effects.