A novel, sensitive DNA hybridization detection protocol, based on DNA-quantum dots nanoconjugates coupled with electrochemical impedance spectroscopy (EIS) detection, is described. The amino-linked ss-DNA probe was covalently immobilized onto a self-assembled mercaptoacetic acid monolayer modified gold electrode; after hybridization with the target ssDNA-CdS nanoconjugates, EIS was used to detect the change of interfacial electron-transfer resistance (Ret) of the redox marker, [Fe(CN)6]4−/3−, from solution to transducer surface. The results showed that when target ssDNA-CdS nanoconjugates hybridized with probe oligonucleotide, a double helix film formed on the electrode, a remarkably increased Ret value was observed. Only complementary DNA sequence had an obvious signal compared with three-base mismatched or non-completely matched sequences under the optimized experimental conditions. Due to having more negative charges, space resistance and the semiconductor property, CdS nanoparticle labels on target DNA could improve the sensitivity to two orders of magnitude when compared with non-CdS tagged DNA sequences.