Abstract: A new pyrrolidine derivative of 2-(2-hydroxy-3-methoxyphenyl)-C60 pyrrolidine derivative (HMP-C60) was synthesized by a 1, 3-dipolar [3+2] cycloaddition reaction, and the chemical structure of the product was characterized by infrared spectroscopy, ultraviolet absorption spectroscopy, elemental analysis and liquid-mass spectrometry. The HMP-C60 was then immobilized on the surface of the glassy carbon electrode by coating. Then, on the basis of the strong coordination between Zr4+ and the oxygen-containing groups, the Zr4+ was used as a bridging reagent to tether probe DNA with the 5'-end modified phosphate group on the surface of the HMP-C60 modified electrode. Thus, an electrochemical DNA sensor based on HMP-C60 modified electrode was constructed. Using [Fe(CN)6]3-/4- as the electroactive probe, the electrochemical characterization of different modified electrodes was carried out, and the performance of the sensors for analyzing the characteristic DNA fragments of cauliflower Mosaic virus (CaMV35S) promoter was investigated by impedance method. The experimental results showed that in the concentration range of 1.0×10-13 ~ 1.0×10-9 mol/L, the electron transfer impedance changes (ΔRet) of the biosensor had a good linear relationship with the logthrim values of the target DNA concentrations (lgCS2), and the detection limit was estimated to be 4.0×10-14 mol/L (S/N=3). The biosensor can effectively identify complementary sequences, base-mismatched sequences and non-complementary sequences, showing a good selectivity.