The invention relates to a preparation method of soluble protein of novel GyV8 gyrovirus VP3. The preparation method comprises the following steps of using a pGEX-6p-1 linear carrier and a primer of a GyV8 virus VP3 gene segment to directly and quickly recombine and clone VP3 in vitro through recombinase ExnaseTMII without enzyme-cut and link up reaction, converting colibacillus coli, and performing IPTG (isopropyl-beta-d-thiogalactoside) inducing expression to realize the fusion soluble expression of GyV8 VP3 protein and GST (glutathione transferase), and obtain purified VP3 soluble protein. The preparation method has the advantages that the GyV8 virus VP3 gene is cloned by the recombinase ExnaseTM II in-vitro recombinant technique without relying on the enzyme cutting site and restriction endonuclease, the cloning process is simplified, and the quick cloning and expression of VP3 gene is realized.