The current invent relates to the construction and application of a three-line breeding system in the Peruvian scallop×bay scallop hybrids. The said three-line consists of a male sterile line, a maintainer line and a restorer line of the bay scallop×Peruvian scallop hybrids. Herein the Peruvian scallop×bay scallop hybrids are hybrids resulted from the eggs of the Peruvian scallops fertilized with sperm of the bay scallops. In constructing the three-line, male sterile individuals are selected from F1 inter-specific hybrid families and continuously backcrossed with sperm of the selling family of the sperm-providing bay scallops of the F1 hybrid to obtain a combination of male sterile line and maintainer line whose hybrid progenies are all male sterile. Then selected individuals from the male sterile line are continuously backcrossed with sperm from the selfing family of a bay scallop until the resulted progenies are all hermaphroditic and exhibit excellent production traits. Commercial male sterile brood stocks are produced by backcrossing the eggs of the male sterile line with the sperm of the maintainer line and commercial Peruvian scallop×bay scallop hybrid spats are produced by backcrossing the eggs of the male sterile line and sperm of the restorer line. This invent establishes the three-line breeding system in animals for the first time and efficiently overcomes the barrier of self-fertilization in the hybridization between hermaphroditic scallops. The yield of the hybrid scallops can be increased by over 50% compared with the bay scallops.