Abstract Leptospirosis is a worldwide zoonosis. Today, serological diagnosis is generally assessed by MAT. We performed ELISA with a synthetic peptide derived from Hap1/lipL32 which is a protein expressed only by pathogenic Leptospira. Repeatability and thresholds were defined with 85 controls sera and 119 hospitalized leptospirosis. The PP-ELISA repeatability and IgM/IgG cut-off values were based on control sera. For these cut-off values, we observed the IgM-PP-ELISA specificity of 89%, whereas it was 100% for the IgG. Then, we compared PP-ELISA and standard MAT results for leptospirosis patients. The concordance rate for IgM-PP-ELISA and MAT was low (43%), whereas it was 85% for IgG-PP-ELISA and MAT. During the first 5 days after hospitalization, PP-ELISA gave positive results in 13 out of 16 patients (81%) whereas 8 out of 14 patients (57%) were positive to MAT. ELISA using Hap1/lipL 32-derived synthetic peptide PP is an earlier serological diagnosis of human leptospirosis than MAT.