By using nested polymerase chain reaction (PCR) composed of three primers that derived from Helicobacter pylori's (Hp) urease gene region, we have not only successfully amplified control Hp and 214 clinic samples but also studied deeply preparation of the sample, composition of PCR reaction liquid and circulatory mental conditions of the PCR. So this method shortens the operation time, simplifies processes and reduces costs. Compared with urease test (54%) and Warthin-Starry silver stained test (50%), ever used the positive rate of PCR is the highest (61%), so our method is more specific, sensitive, simple, rapid and accurate, and could be applied to the routine clinical detection.