In this work, a novel electrochemical sensor was prepared based on self-assembled homocysteine molecular layer on the surface of a three-dimensional (3D) nanoporous gold leaf (NPGL) modified gold electrode (HCys/NPGL/GE) and applied for trace measurement of Pb(II) by differential pulse anodic stripping voltammetry (DPASV). NPGL is introduced in order to enhance electron transport efficiency and surface area of sensor, while homocysteine as an adapter on NPGL surface can capture Pb(II) and further elevate sensitivity and selectivity in detecting Pb(II). The DPASV response of Pb (II) at the HCys/NPGL/GE was about four and three times larger than that at HCys/GE and NPGL/GE, respectively. Factors influencing determination of Pb(II) including pH value, deposition potential and deposition time were investigated. Under optimal conditions, the electrochemical sensor exhibited a wide linearity range from 1 mu g/L to 200 mu g/L Pb(II) with a detection limit of 0.1 mu g/L (S/N = 3). The detection limit is much lower than 10 pg/L, which is the guideline value in drinking water given by the World Health Organization. Application of the established sensor in detecting Pb(II) in routine samples, including Chinese herb (Radix Scutellariae), blood and waste water, has been realized, and the results agreed well with those obtained by hydride generation atomic fluorescence spectrometry. (C) 2015 Elsevier B.V. All rights reserved.