A gold nanoparticles modified carbon paste electrode(GNPs/CPE) was prepared,and the electrochemical characteristics of methotrexate(MTX) at GNPs/CPE were studied by cyclic voltammetric method.A novel electrochemical method for the determination of MTX at GNPs/CPE was established and its interaction with herring sperm DNA(hsDNA) was investigated by linear sweep voltammetry(LSV) and square wave voltammetry(SWV).The results indicated that there was a good linear relationship between anodic peak current and MTX concentration in the range of 0.5-10.0 μmol?L-1,and the detection limit(S/N=3) was 0.2 μmol?L-1.The relative standard derivations(n=11) was 4.7% for 3.0 μmol?L-1 MTX.The method was applied in the determination of MTX in the injection samples with satisfactory results.The results also showed that,in the presence of various concentrations of hsDNA,the redox peak potential of methotrexate shifted positively and its peak current was decreased,which indicated that MTX could react with hsDNA and form an electrochemical inactive compound.The binding equilibrium constant and binding ratio were calculated to be 4.6×105 L?μmol-1 and 2∶1,respectively.