Background: Canine Leptospirosis is a life-threatening disease and zoonosis. Usually, PCR assay is carried out for early diagnosis but requires a thermal cycler and post-PCR procedures. This limits its use in resource-limited areas. Hence, the isothermal amplification of nucleic acid by recombinase polymerase amplification assay was developed as a versatile alternative for the diagnosis of canine leptospirosis in this study. Methods: The RPA assay to detect Leptospira DNA was optimized with Leptospira reference strains and its performance characteristics such as analytical, diagnostics and reproducibility were assessed. Result: The limit of detection of RPA assay was estimated as 102 copies of genomic DNA and specific to amplify the pathogenic Leptospira. Out of 150 dog samples screened, Leptospira DNA was detected in 64 (42.6%) by RPA assay and 67 (44.6%) by PCR. The diagnostic sensitivity and specificity of the RPA assay were 92.5% and 97.59% respectively. The RPA assay has a good diagnostic agreement with a kappa value of 0.905. The reproducibility assessment with the third-party testing laboratory revealed a better agreement with a kappa value of 0.81. The simplicity, rapid and less expensive enable this assay to perform at resource-limited laboratories or point-of-care testing.