Abstract: Objective To compare and evaluate the five detection methods of Clostridium difficile. Methods The five methods included PCR, real-time PCR, enzyme-linked immunosorbent assay (ELISA), immunochromatographic assay (GICA) and cycloserin-cefoxitin-fructose agar (CCFA) culture. The sensitivity, specificity, positive predictive value, negative predictive value and Kappa value of the five methods were compared. Among the 125 samples, 11 were CCFA culture positive, 12 were tcdB in PCR, 12 were tcdB positive in real time PCR, 50 were positive in ELISA and 25 were positive in GICA, the differences in positive rate of 4 methods were significant (2=0.288, P=0.962). The specificities were 95.61%, 95.61%, 62.28% and 83.33% respectively, the differences were significant (2=63.597, P0.000 1). Conclusion PCR, which had highest sensitivity and specificity, but had low cost, is appropriate to be used in epidemiological research and clinical diagnosis. ELISA and GICA can be used in initial screening and auxiliary diagnosis.