Abstract DNA hybridization and enzymatic digestion for the detection of mutation was investigated on the gold nanoparticles–calf thymus DNA (AuNPs–ctDNA) modified glassy carbon electrode (GCE). The thiol modified probe oligonucleotides (SH–ssDNA) were assembled on the surface of AuNPs–ctDNA modified GCE. The electrochemical response of the electrode was measured by differential pulse voltammetry and cyclic voltammetry. Methylene blue (MB) was used as the electroactive indicator. AuNPs were then dispersed effectively on the GCE surface in the presence of ct–DNA. When hybridization occurred, a decrease in the signal of MB current was observed. The modified electrode was used for the detection of mutations during the enzymatic digestion reaction in DNA. During this reaction, an increase in the signal of MB current was observed. So, the modified SH–ssDNA had a higher electrochemical response on the AuNPs–ctDNA/GCE because of the strong affinity of MB for guanine residues in it. The electrochemical detection of restriction enzyme digestion can provide a simple and practical method for observing single-base mismatches that can help in distinguishing mismatch sequences of DNA from the complementary ones.