Opisthorchis viverrini, a food-borne trematode parasite endemic in the lower Mekong countries, is conventionally diagnosed by stool examination. However, parasitological stool-based diagnosis can be unreliable in light infections. The goal of this study was to develop the immunodiagnosis of opisthorchiasis using cathepsin F cysteine protease of O. viverrini in both indirect and sandwich ELISA assays. A recombinant O. viverrini cathepsin F (rOv-CF) of 40 kDa was expressed in E. coli strain BL21 (DE3), affinity purified, and deployed in ELISA assays. Human sera from 272 cases were investigated by indirect rOv-CF-based ELISA. Positive antibody response to rOv-CF was found in 137 out of 272 cases (50.37 %) using a cutoff OD (0.400) determined by ROC analysis. In comparison to parasitological stool examined for fluke eggs, the gold standard, the rOv-CF indirect ELISA showed a sensitivity and specificity of 62.1 and 84.05 %, respectively. Serum antibody levels correlated well with egg counts per gram feces (EPG) (P