Leptospirosis is a widespread zoonotic disease that affects all mammals, including humans, in different parts of the world. Clinical recognition of leptospirosis is challenging, and the definitive serologic diagnosis assay, the microscopic agglutination test (MAT), is time-consuming and difficult to conduct. In this study, an indirect immunoperoxidase (IIP) test to detect Leptospira-specific antibodies in human serum samples was developed. The efficacy of the IIP was compared with the indirect immunofluorescent assay (IFA) and MAT. A total of 368 human serum samples were analyzed by MAT, IFA, and IIP. Using a MAT titer of > or = 1:100 as the gold standard, the sensitivities for the detection of Leptospiral antibodies at a titer of 1:200 were 94.7% by IFA and 93.6% by IIP; specificities were 95.3% by IFA and 94.9% by IIP; and accuracies were 95.1% by IFA and 94.6% by IIP. With a titer of 1:400, the sensitivity, specificity, and accuracy were 86.2%, 98.9%, and 95.7% by IFA, respectively; whereas, for the IIP, the sensitivity was 85.1%, specificity 98.5%, and accuracy 95.1%. A further evaluation of this test with 80 unknown-febrile-disease sera was also included. We found that the sensitivity and specificity of this test were 100% and 76.8%, respectively. Therefore, the IIP test is a potentially valuable tool for the diagnosis of leptospirosis.