Opisthorchis viverrini is an important food-borne trematode in Southeast Asia. The infection causes significant morbidity in terms of hepatobiliary diseases and cholangiocarcinoma. The aim of this study was to improve the sensitivity of the PCR-based diagnosis of O. viverrini infection. A new fecal DNA extraction protocol for the detection of O. viverrini DNA using cetyltrimethyl-ammoniumbromide to remove PCR inhibitor was used and compared with the commercial stool kit method. The sensitivity of the new test was 79.3%, compared with the 44.8% of the previous method (P < 0.01). PCR-positive tests identified several cases judged parasite negative by the parasitological method (28.6%), indicating the new test's advantage in the diagnosis of individuals with light infections.