Using three strains of Fusarium oxysporum f.sp.cucumarinum, twenty three other Fusarium isolates, thirty three comparison strains from cucumber rhizosphere as materials, we used PCR-RFLP and Nested-PCR for detecting Fusarium oxysporum f.sp.cucumarinum on base of total DNA chemical cracking method. It showed that PCR-RFLP could not differenciate different species of Fusarium. Nested-PCR is special for Fusarium oxysporum f.sp.cucumarinum. PCR-RFLP and Nested-PCR detected successfully the pathogen in diseased cucumber plants inoculated with Fusarium oxysporum f.sp.cucumarinum. The two programs both can be used to detect Fusarium oxysporum f.sp.cucumarinum in cucumber tissues without eyeable symptoms. PCR-RFLP could detect Fusarium oxysporum f.sp.cucumarinum in the inoculated tissues of susceptible cucumber plants on 3rd day after inoculation, but Nested-PCR could detect the pathogens on the 5th day after inoculation. It indicates that PCR-RFLP is more sensitive than Nested-PCR in detection of Fusarium oxysporum f.sp.cucumarinum.