This paper described how a inorganic–organic hybrids electrochemical sensor, a three-dimensional l-cysteine self-assembled monolayers (SAM) functionalized gold nanoparticles/single-walled carbon nanotubes/glassy carbon electrode (l-cys/AuNPs/SWCNTs/GCE), was applied to improve the performance of detecting trace Cu(II) in stripping voltammetry. The AuNPs/SWCNTs nanohybrid has a three-dimensional porous nanostructure with large active surface area, and the l-cysteine adapters on AuNPs/SWCNTs surface can greatly enhance the sensitivity and selectivity in detecting Cu(II). The differential pulse anodic stripping voltammetry (DPASV) response of the Cu(II) at the l-cys/AuNPs/SWCNTs/GCE was ca. 6.7 and 6.5 times larger than that at the AuNPs/SWCNTs/GCE and l-cys/AuNPs/GCE without SWCNTs, respectively. The sensor demonstrated a wide linear response range and a lower detection limit of 0.02nM with a signal-to-noise of 3 using 3min of preconcentration time. The interference experiments showed that Ag(I), Pb(II), Cd(II) and Hg(II) had little influence on Cu(II) signal. The high sensitivity and excellent selectivity in contrast to the values reported previously in the area of electrochemical Cu(II) detection, demonstrated the analytical performance of the as-prepared sensor toward Cu(II) was superior to the existing electrodes. The as-prepared sensor was further applied to determine the Cu(II) in the real environmental water sample, and the results agreed satisfactorily with the certified values.