The invention provides a pair of specific primers used for PCR detection of Aphelenchoides besseyi and an application method thereof. The critical innovation of the method lies in that the newly-designed specific primers for Aphelenchoides besseyi can be combined with reported universal primers for ITS of ribosome of plant-parasitic nematodes so as to establish a novel duplex PCR detection method for rapid identification of Aphelenchoides besseyi. The method provided by the invention is highly efficient and stable, reaches a sensitivity level of 1/10 nematode DNAs and can be promoted and applied in quarantine and identification of ports and agricultural and forestry departments in China.