Objective To explore the interference of recent use of antibiotics in the sensitivities of different methods for the detection of Helicobacter pylori (Hp) in children. Methods From June 2015 to December 2017, children who were highly suspected of Hp infection and required gastroscopy were divided into the antibiotic group and the control group according to the recent use of antibiotics, with 200 cases in each group. The use of antibiotics of children in the antibiotic group was analyzed. The children in the two groups completed five methods of Hp detection, including rapid urease test (RUT), 13C-urea breath test (13C-UBT), pathological Warthin-Starry staining, colloidal gold method for measuring Hp immunoglobulin M (IgM) antibody, and enzyme linked immunosorbent assay method for measuring Hp stool antigen (HpSA). Comparative analyses of data were done. Results The positive rate of RUT was 36.5% in the antibiotic group and 75.0% in the control group (P<0.05). The positive rate and value of13C-UBT were 59.0% and 13.6±4.7 in the antibiotic group, and 78.0% and 41.7±6.6 in the control group (P<0.05). The positive rate of pathological biopsy was 48.0% in the antibiotic group and 68.0% in the control group (P<0.05), and L-forms were found in 80.9% of the Hp-positive specimens in the antibiotic group. The positive rate of IgM antibody was 54.5% in the antibiotic group and 65.5% in the control group (P<0.05). The positive rate of HpSA was 38.0% in the antibiotic group and 69.0% in the control group (P<0.05). In the antibiotic group, only 12.5% of the children used antibiotics for eradication of Hp and 87.5% for non Hp eradication. Conclusions Under the interference of antibiotics use, the sensitivity of 13C-UBT is the highest among the five methods for Hp detection, and the sensitivities of RUT and HpSA are very low. For children who could not cooperate with 13C-UBT, the sensitivity of IgM detection is relatively high. For children who need gastroscopy, prolonged chromogenic time for RUT and multi-point pathological biopsy can reduce misdiagnosis.