Resistance to clarithromycin (CLA) and levofloxacin (LFX) of is increasing in severity, and successful eradication is essential. Presently, the eradication success rate has greatly declined, leaving a large number of patients with previous treatment histories. To investigate secondary resistance rates, explore risk factors for antibiotic resistance, and assess the efficacy of susceptibility-guided therapy. We recruited 154 subjects positive for Urea Breath Test who attended The First Affiliated Hospital of China Medical University between July 2022 and April 2023. Participants underwent a string test after an overnight fast. The gastric juice was obtained and transferred to vials containing storage solution. Subsequently, DNA extraction and the specific DNA amplification were performed using quantitative polymerase chain reaction (qPCR). Demographic information was also analyzed as part of the study. Based on these results, the participants were administered susceptibility-guided treatment. Efficacy was compared with that of the empiric treatment group. A total of 132 individuals tested positive for the gene by qPCR technique. CLA resistance rate reached a high level of 82.6% ( = 109), LFX resistance rate was 69.7% ( = 92) and dual resistance was 62.1% ( = 82). Gastric symptoms odds ratio (OR) = 2.782; 95% confidence interval (95%CI): 1.076-7.194; = 0.035 and rural residence (OR = 5.152; 95%CI: 1.407-18.861; = 0.013) were independent risk factors for secondary resistance to CLA and LFX, respectively. A total of 102 and 100 individuals received susceptibility-guided therapies and empiric treatment, respectively. The antibiotic susceptibility-guided treatment and empiric treatment groups achieved successful eradication rates of 75.5% (77/102) and 59.0% (59/411) by the intention-to-treat (ITT) analysis and 90.6% (77/85) and 70.2% (59/84) by the per-protocol (PP) analysis, respectively. The eradication rates of these two treatment strategies were significantly different in both ITT ( = 0.001) and PP ( = 0.012) analyses. presented high secondary resistance rates to CLA and LFX. For patients with previous treatment failures, treatments should be guided by antibiotic susceptibility tests or regional antibiotic resistance profile.