Background and Objectives: Diphtheria is a potentially fatal disease caused by toxigenic bacterial infection, particularly from Corynebacterium diphtheriae (C. diphtheriae) . Isolation of C. diphtheriae is technically lacking in sensitivity, and Elek’s test to detect toxin production has several difficulties associated with its application. Duplex real-time PCR to throat swab of suspected diphtheria patients can detect both bacteria and toxin-encoding genes simultaneously, faster, with higher sensitivity and specificity. Materials and Methods: A total of 89 consecutive throat swabs from suspected diphtheria patients were collected from Sulianti Saroso Infectious Disease Hospital, Jakarta, during 2018 to 2019. Two pairs of primers and probes, targeting the rpoB gene of C. diphtheriae and the A-subunit of the diphtheria toxin gene, were used in this study. Parameters including annealing temperature, concentration of primers and probes, inhibitors, cross-reaction and detection limit were all optimized. Elek’s toxigenicity test and clinical data were analyzed for comparison. Results: The optimum annealing temperature was 55°C. The concentrations of Cd primer, Tox primer, Cd probe and Tox probe were 0.4, 0.6, 0.5 and 0.625 µM, respectively. DNA elution and template volumes were 50 µL and 5 µL. The detection limit was 2 CFU/mL. No cross-reaction with other microorganisms was observed. Of the 89 samples, duplex real-time PCR gave better results than the standard test, with 19 (21.3%) and 10 (11.2%) patients diagnosed with diphtheria, respectively. Conclusion: Duplex real-time PCR increases the rate of laboratory diagnosis of diphtheria, compared to the standard method to detect potentially toxigenic C. diphtheriae .