A label free electrochemical biosensor was developed for the detection of p53 tumor suppressor gene using hairpin DNA as nucleic acid probe and RuHex as hybridization indicator,based on the specific recognition of hairpin DNA and the electrostatic interaction between［Ru（NH3）6］3+ and DNA. The experimental results indicated that this electrochemical sensor can sensitively detect target DNA in 10 μmol/L RuHex solution with a detection limit of 2.5 nmol/L. According to the difference of the electrochemical signals,the sensor could discriminate the complete complementary DNA from single-mutation DNA and the random DNA. The hybridization efficiency of hairpin DNA and the target sequences with different mutation sites was investigated and the results showed that the mutate sites affected DNA hybridization efficiency. The prepared sensor showed the advantages of sensitivity,selectivity,label free and simplicity.