Helicobacter pylori (H. pylori) infection has long been a significant global public health concern, with antibiotic resistance becoming increasingly severe. Gastric fluid qPCR detection can provide information on H. pylori infection and antibiotic resistance. This study compares a series of diagnostic methods among 300 participants to reveal best practices for detecting H. pylori infection and/or profiling antibiotic resistance, including urea breath test (UBT), rapid urease test (RUT), gastric mucosa qPCR (GM-qPCR), and the novel gastric fluid qPCR (GF-qPCR). The infection rates detected by UBT and RUT were 32.33% and 23.33%, respectively. Through qPCR analysis, the infection rates in gastric fluid and gastric mucosal were 27.67% and 25.33%. When comparing three methods of detecting H. pylori using the UBT as the reference standard, it was found that the sensitivity, specificity, positive and negative predictive values, and accuracy of gastric fluid qPCR performed the best. The consistency of the four methods was assessed using the Kappa value, which yielded a value of 0.792. Additionally, 45 individuals showed inconsistent results. When using qPCR to detect antibiotic resistance in gastric fluid and gastric mucosal samples, we observed resistance rates of 42.17% (35/83) for clarithromycin and 49.40% (41/83) for levofloxacin in gastric fluid samples. In contrast, resistance rates in gastric mucosal tissue samples were significantly lower at 16.87% (14/76) for clarithromycin and 22.37% (17/76) for levofloxacin. The GF-qPCR method is a promising technique for detecting H. pylori infection and antibiotic resistance, with the potential for providing guided treatment plans.