The invention relates to a method for authenticating oyster population, in particular to a novel method for authenticating Pacific oyster population, comprising the steps of sample collection, genome extraction, primer design, PCR amplification, and EST-SSRs specific site authentication. In the step of sample collection, sample individuals from different populations of ostrea cucullata, Pacific oyster and crassostrea rivularis are collected; in the step of genome extraction, genomes of a plurality of samples of each oyster population are extracted; in the step of primer design, EST-SSRs primers are designed according to an expressed sequence tag (EST) sequence containing microsatellites; in the step of PCR amplification, the EST-SSRs primers are used to carry out the PCR amplification to obtain the PCR product with a target segment; and in the step of EST-SSRs specific site authentication, agarose gel electrophoresis is firstly carried out on the PCR product to obtain the PCR product with clear and stable bands, and then polyacrylamide gelelectrophoresis is carried out to detect polymorphic sites so as to authenticate the allele Pacific oyster population having the allele sites with specific EST microsatellite sequence. In the invention, an EST-SSRs molecule marker technology is utilized, the primers are reasonably designed, and two-step electrophoresis detections are adopted so as to achieve the purpose of authenticating the Pacific oyster population. The invention has the advantages of simple method, time saving and accurate analysis.