The invention discloses a method for testing the distinctness, uniformity and stability of a pure-line rice new variety. The method specifically comprises the following steps: obtaining variation points; determining a testing area of the rice variety to be tested; establishing a database; after determining the sampling amount, randomly sampling, mixing samples, and extracting DNA in the mixed sample; preparing primers; amplifying the DNA in the mixed sample by using the primers, and establishing a high-throughput sequencing library by using the product generated after amplification; performing high-throughput sequencing on the high-throughput sequencing library, so as to obtain a sequencing fragment group; analyzing the sequencing fragment group, so as to obtain the genotype of the rice variety to be tested and the genotype of an abnormal plant; obtaining similar varieties, variation points and variation point rate through comparison; after obtaining an abnormal plant variety through comparing the genotype of the abnormal plant and genotypes in the database, computing the abnormal plant rate; judging the distinctness, uniformity and stability of the rice variety to be tested according to the variation points, variation point rate and abnormal plant rate. The method can be used for accurately and completely judging the distinctness, uniformity and stability of the plant variety to be tested.