Abstract: Grapholitha molesta is a common internal fruit feeders from orchard in China, and is very similar to other pear pests in the external morphology. It is difficult to distinguish only by morphological characteristics, a real-time fluorescent PCR detection method should be established for identification at the gene level. The specificity of fluorescent probe was designed and verified through sample nucleic acid extraction and quality control, and a real-time fluorescent quantitative PCR detection method for Grapholitha molesta was developed by further optimizing reaction conditions and reaction systems, combined with specificity analysis, sensitivity comparison and blind sample testing. The results showed that the method had strong specificity, good repeatability（the coefficient of variation between repeats was0.055～0.359）, high reliability（R2value of standard curve was 0.991）, and low detection limit（the minimum detection limit was 3 pg/μL）, the results of blind sample test were consistent with morphological identification results, which could meet the needs of daily inspection and quarantine. This method is simple and easy to operate, with short detection time limit（about 3 h） and high flux, which is especially suitable for sampling inspection in large quantities, and can be applied to the quarantine of Grapholitha molesta.