Abstract: BACKGROUND: Screening for transfusion-transmitted infections (TTIs) is an important step for the safety of blood transfusion. Major TTIs for which screening is done are hepatitis B surface antigen, antibody to hepatitis B virus, hepatitis C virus, human immunodeficiency virus, syphilis, and malaria. The aim of this study was to compare the diagnostic accuracy of treponemal-specific chemiluminescence test and rapid card test for the screening of syphilis by taking Treponema pallidum hemagglutination assay (TPHA) as a gold standard test. MATERIALS AND METHODS: This was a cross-sectional study. All the routine blood donors were screened according to the National Blood Transfusion Council guidelines. One hundred and twenty syphilis serology-positive samples were obtained either by rapid card test immunochromatographic test/chemiluminescence assay or both. These samples were further tested by TPHA (gold standard test). Statistical analysis was done using the Mcnemar test, DeLong et al. test, and Chi-square test P < 0.05 was considered statistically significant. RESULTS: The sensitivity and specificity of both chemiluminescence and rapid card test were comparable (P < 0.0001) and the diagnostic accuracy of chemiluminescence and rapid card test was 95.83% and 94.58%, respectively. CONCLUSION: Both chemiluminescence and rapid card tests show comparable performance, so it is concluded to use rapid card test as a reliable backup in remote areas, peripheral blood bank setup, and in emergency situations where timesaving is lifesaving.