The invention discloses a method for preparing Acaudina leucoprocta collagen oligopeptide through hydrolysis with combination of collagenase and novel microbial protease. The method comprises: pretreatment, collagen extraction, fermentation, microbial protease purification, enzymolysis, microbial protease enzymolysis, and purification. According to the present invention, during the enzymolysis, the dual-frequency ultrasonic wave assisted enzymolysis is used, such that the energy barrier between the ground state and the active state of the hydrolysis reaction can be reduced, and the Ea value and the KM value of the enzymolysis reaction can be reduced so as to increase the affinity between the matrix and the enzyme, greatly increase the enzymatic reaction rate, and save the time cost and thelabor cost; after the Acaudina leucoprocta collagen is hydrolyzed by combining the collagenase and the novel microbial protease, the collagen is hydrolyzed into the amino acids with low molecular weight and the oligopeptide with low molecular weight so as to be easily absorbed by the body and easily provide the medical health effects of the Acaudina leucoprocta collagen oligopeptide; and the obtained product is the nutrition health substance with characteristics of easy absorption, high nutrition and no toxic-side effect.