The invention discloses a screening method of high-yield strains of nattokinase. According to the method, rapid and accurate screening of the high-yield strains of nattokinase is realized with technologies of high-throughput screening, OD (optical density) value rapid detection and the like by means of design and application of a selective culture medium on the basis of physical and chemical characteristics of nattokinase and growth and metabolism characteristics of bacillus natto. Compared with a traditional method, the screening method has the advantages of clear direction, precise target, small workload, short time, high efficiency, high success rate and the like, so that not only are defects of large workload, low efficiency, high screening randomness and poor targeted performance of the screening method overcome, but also the screening cycle is obviously shortened; compared with an advanced gene site-directed mutation screening method, targeted performance and directionality of screening are not reduced, a large quantity of manpower, material resources, financial resources and the like are also saved, and meanwhile, the screening difficulty is greatly reduced. The method is simple to operate, convenient and fast and can be widely applied to rapid screening of serine protease producing strains.