<p>The rising resistance of pathogens has increasingly complicated antibiotic treatments, leading to prolonged therapy durations and the potential for inadequate disease management. Current clinical methods (e.g., culture-based AST, conventional PCR, Sanger sequencing) for assessing microbial resistance are hampered by lengthy processing times, limited sensitivity, or high costs. In this study, we developed a multiplex quantitative polymerase chain reaction (qPCR) method using custom-designed universal locked nucleic acid (LNA) probes. This approach eliminates the need for target-specific fluorescent probes required in conventional qPCR assays, thereby offering broad applicability and reduced development costs. Using this method, a multiplex qPCR assay consisting of four tubes of four-plex reaction for the detection of 16 common antibiotic resistance genes (ARGs) was developed. These ARGs correspond to seven types of commonly used antibiotics and show high concordance with phenotypic resistance profiles. The established multiplex system demonstrated robust amplification performance, with a detection limit of 25 copies/μL for all targets. The analytical performance of this assay was validated in 172 samples from patients with severe pneumonia, achieving complete concordance with Sanger sequencing. Overall, this study provides an accurate, cost-effective, and rapid solution for detecting microbial resistance, enabling timely informed selection of antibiotics in clinical practice.</p>