2,3-Dimercaptosuccinic acid (DMSA) and its complex Nickel(Ⅱ) 2,3-Dimercaptosuccinate (DMSA-Ni(Ⅱ)) were assembled on the electrochemically activated Au electrode and the modified films were investigated by ex-situ SERS, SEM and electro-reduction. The coordination ratio (1∶2) and stability constant (4.716×107) of DMSA and Nickel(Ⅱ) were firstly confirmed by UV. Then, the SERS spectra showed that DMSA was absorbed on the surface of activated Au and the coordinate bond of DMSA-Ni(Ⅱ) was existed as S—Ni—O. Meanwhile, two reduction peaks of DMSA-Ni(Ⅱ) on Au electrode were presented at negative potential. The first peak (from positive to negative values) was related to the RS—Au group which was produced by the mutual absorption of DMSA and Au. The second reduction peak was related to Nickel(Ⅱ). Finally, it was further confirmed by calculating the surface coverage and comparison of molecular area. And it was found that the molecular arrangement of DMSA-Ni(Ⅱ) on Au electrode generated as monolayer which was dispersed or connected partly instead of concentrated or overlapped.