Fusarium oxysporum is one of the most important phytopathogens and cause Fusarium wilt disease in cucumber,watermelon and melon,etc.In this study,a pair of species-specific primers Fc-1 and Fc-2 was synthesized based on differences in internal transcribed spacer sequences of Fusarium genus.With the primers,a specific 315 bp PCR product was amplified from five F.oxysporum isolates isolated from cucumber,watermelon and melon,infected cucumber and watermelon tissues,while no product was obtained from other fourteen fungi,healthy cucumber and watermelon tissues.The detection sensitivity is 100 fg for genomic DNA of F.oxysporum and 1 000 spores/g soil for the soil pathogens.In contrast,the nested PCR with two pairs of primers(ITS1/ITS4 and Fc-1 /Fc-2) increased the sensitivity by 100-fold.In addition,one-step PCR could also detect F.oxysporum in symptomless cucumber root of 7 dpi(days post inoculation) and in infected cucumber and watermelon tissues at the early stage of disease development.Therefore,the developed PCR-based method enabled rapid,sensitive and reliable detection of F.oxysporum.It also provides the detection method for early monitoring and diagnosis of the pathogen as well as the plant disease management guidance. Fusarium oxysporum is one of the most important phytopathogens and cause Fusarium wilt disease in cucumber, watermelon and melon, etc. In this study, a pair of species-specific primers Fc-1 and Fc-2 was synthesized based on differences in internal transcribed spacer sequences of Fusarium genus. The primers, a specific 315 bp PCR product was amplified from five F.oxysporum isolates isolated from cucumber, watermelon and melon, infected cucumber and watermelon tissues, while no product was obtained from other fourteen fungi, healthy cucumber and watermelon tissues. The detection sensitivity is 100 fg for genomic DNA of F.oxysporum and 1 000 spores / g soil for the soil pathogens. In contrast, the nested PCR with two pairs of primers (ITS1 / ITS4 and Fc- 1 / Fc- 2) increased the sensitivity by 100- fold. Addition, one-step PCR could also detect F. oxysporum in symptomless cucumber root of 7 dpi (days post inoculation) and in infected cucumber and watermelon tissues at the early stage of disease development . Prior, the developed PCR-based method enabled rapid, sensitive and reliable detection of F. oxysporum. It also provides the detection method for early monitoring and diagnosis of the pathogen as well as the plant disease management guidance.