In this paper, we demonstrated the fabrication of high active and high sensitive Au nanoparticles by laser ablation in liquid (LAL) method, and their application in electrochemical detection of heavy metal ions. First, LAL method are used to fabricate Au nanoparticles in water in a clean way. Second, the Au nanoparticles were assembled onto the surface of the glassy carbon (GC) electrode by an electrophoretic deposition method to form an AuNPs/GC electrode for electrochemical characterization and detection. Through differential pulse anodic stripping voltammetry method, it shows that the AuNPs/GC electrode could be used for the simultaneous and selective electrochemical detection of Cd(2+), Pb(2+), Cu(2+), and Hg(2+). By studying the influence of test conditions to optimize the electrochemical detection, we can detect Cd(2+), Pb(2+), Cu(2+), and Hg(2+) simultaneously with a low concentration of 3 × 10(-7) M in the experiments.