The invention discloses a preparation method of kiwi fruit enzyme with high anti-oxidation activity. A formula is characterized in that fresh kiwi fruits are adopted as raw materials, fruit skin is reserved for pulping after washing and deburring the kiwi fruits, and enzymolysis is carried out on kiwi fruit pulp by adding a composite enzyme preparation (which is prepared from pectinase, cellulose, hemicellulase, protease and diastase), so that the juice yield and the production efficiency are improved; then, a multi-strain symbiotic fermentation technology and a grading inoculation process are adopted, and fermentation and ageing are carried out on a product by sequentially adding saccharomycetes, acetic acid bacteria and lactic acid bacteria; finally, the purpose of removing bacteria is achieved through the technologies of kieselguhr filter, membrane filter and the like, so that continuous fermentation of the product is ended, the stability of the shelf life is ensured, and meanwhile, the activity of active ingredients and enzyme is maximum kept. The enzyme prepared through the preparation method has the characteristics of high anti-oxidation activity, richness in enzymes and high enzyme activity; the appearance is clear and transparent, the flavor is sour, sweet and gentle, and the fruity fragrance is elegant.