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Introduction: The World Health Organisation (WHO) estimated the annual incidence of leptospirosis as 0.1–1 case per 100,000 population in temperate and non-endemic areas, while it is 10–100 cases per 100,000 population in humid, tropical and endemic areas. The clinical features may vary from asymptomatic to lethal disease and are based on the virulence of the infective serovar, infective dose, age, occupation and immunological status of the host. Laboratory diagnosis is pivotal because of the diverse clinical manifestations of the disease, which are mainly achieved by isolation of the organism by culture or by demonstration of a rise in specific serum antibody. This study aims to compare the performance of polymerase chain reaction (PCR) and Immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) with the gold standard microscopic agglutination test (MAT) in diagnosing human leptospirosis. Methodology: The present cross-sectional study was conducted in the Department of Microbiology of a tertiary care centre for six months from July 2022 to December 2022 after collecting 100 serum samples from suspected leptospirosis patients. Results: Diagnosis of leptospirosis using PCR, MAT and IgM ELISA was 6%, 9% and 24%, respectively. Conclusion: Both ELISA and MAT can be done if a single sample is available. PCR can be used as a complementary test in the early phase of infection, especially when the antibody detection is negative.