In the condition of 1.24 mmol/L EDTANa2, 16.7 mmol/L NaCl and 0.17 mmol/L Tris, the substrate chain of double-stranded DNA (dsDNA) could be cracked by Pb2+ to release single-stranded DNA (ssDNA) that adsorb onto AuPd nanoparticle (AuPdNP) and form stable AuPdNP-ssDNA, but the dsDNA can not protect AuPdNP that were aggregated to big AuPdNP aggregations (AuPdNPA) under the action of NaCl. The AuPdNP-ssDNA and AuPdNPA could be separated by centrifugation. With the concentration of Pb2+ increased, the released ssDNA increased, the AuPdNP-ssDNA in centrifugation solution increased and the catalytic effect enhanced on the fluorescence quenching reaction of Rhodamine 6G (Rh6G) and NaH2PO2, which led the fluorescence intensity at 552nm to decrease. The decreased fluorescence intensity (ΔF552nm) was linear to the concentration of Pb2+ in the range of 0.33-8.00 nmol/L, a detection limit of 0.21 nmol/L. The proposed method was applied to detect Pb2+ in water samples, with satisfactory results.