Abstract: Black spot is one of the most severe pear diseases. Establishing a PCR detection method is important for early diagnosis and port plant quarantine. In this study, the ITS sequence of Alternaria alternata and other 19 different fungi were analyzed and compared, and a pair of specific primers were designed which could specifically amplify a 398 bp fragment from Alternaria alternata, but not from other tested fungi. In the sensitivity test, the detection limit of the bacteria genomic DNA were 10 pg/μL, Alternaria alternata was detected in both diseased pear fruit with symptoms of black spot or inoculated pear fruit by Alternaria alternata. Therefore, this pair of primers could be applied to early diagnosis and PCR detection of pear black spot. The study provided useful technical support for controlling this disease by fungicides for farmers.