Abstract: Objective To evaluate the diagnostic efficacy of real-time polymerase chain reaction (q-PCR) for Clostridium difficile infection (CDI) in comparison with routine culture and enzyme-linked fluorescent spectroscopy-based aprroaches. Methods Stool samples were collected from suspected CDI cases in General Hospital of Guangzhou Military Command of PLA between May and December in 2016. All the samples were examined with 3 methods, namely enzyme-linked fluorescent spectroscopy for detecting Clostridium difficile toxin A/B (CDAB), detection of glutamate dehydrogenase (GDH), and q-PCR for amplification of Clostridium difficile-specific gene tpi and toxin gene (tcdA/tcdB), with the results of fecal culture as the reference for evaluating the diagnostic efficacy of the 3 methods. Results Of the total of 70 fecal samples, 13 (18.57%) were found to be positive for Clostridium difficile, including toxin-producing strains in 6 (8.57%) samples. The sensitivity, specificity, positive predictive value, negative predictive value and diagnostic coincidence rate of q-PCR for tpi were 92.31%, 91.23%, 70.59%, 98.11% and 91.43%, respectively, which were significantly higher than those of GDH test (84.62%, 84.21%, 55.00%, 96.00%, and 84.29%, respectively; χ2=24.881, P<0.001). The sensitivity of q-PCR for tcdA/cdB was significantly higher than that of enzyme-linked fluorescent spectroscopy for CDAB in detecting CDI (66.67% vs 33.33%; χ2=35.918, P<0.001). Conclusion Both CDAB detection and q-PCR have a high specificity in detecting CDI, but GDH detection has a good sensitivity, and all these 3 methods have a high negative predictive value. Compared with other detection methods, amplification of tpi and tcdA/tcdB using q-PCR allows more rapid, sensitive and specific detection of CDI.