Abstract: Patulin is the major mycotoxin in apple juice produced in apple juice processing by penicillium expansum, rapid detection of penicillium expansum is of great significance for controlling the fungi and ensuring apple juice quality. Based on isoepoxydon dehydrogenase (primer ISO) and polygalacturonase (primer POL) respectively, two pairs of primers were used to amplify DNA from penicillium expansum and compare the specificity of penicillium expansum DNA amplification. It has been proven that primer pairs(ISO) have poor specificity, while primer pairs(POL) have high specificity and only 404 bp DNA fragments were amplified. It has also been shown from DNA serial dilution amplification sensitivity that the amplification sensitivity of DNA serial dilution from plate culture has at least one fold level higher than that from liquid culture. DNA concentration determination has also proven that DNA concentration from plate culture is much higher than that from liquid culture. The POL primers also had a high amplification specificity of DNA from penicillium expansum inoculated apples and juice, while there were difference of DNA amplification strongness from different apple species. Apple DNA had no interference with amplification of penicillium expansum DNA