LipL32 is not only the most abundant protein of the outer membrane but also an immunodominant antigen that is highly conserved in pathogenic Leptospira. Recombinant LipL32 (rLipL32) purified under natural conditions was adapted to immunize BALB/c mice for developing monoclonal antibodies (mAbs). Twenty-nine mAbs against eight epitopes were produced and characterized. The capture and detection of antibodies were selected using a one-by-one pairing method with biotin-conjugated mAbs. To evaluate the sensitivity and specificity of the mAb pairs, antigen capture enzyme-linked immunosorbent assay (ELISA) was applied to detect antigens extracted from 15 pathogenic Leptospira strains and from other pathogenic bacteria. One pair of mAbs was selected and the detection capability of rLipL32 by ELISA was found to be approximately 1 ng/ml. mAbs produced with rLipL32 purified under natural conditions may be promising in the further detection of leptospiral antigens.