In this paper, a novel sensitive biosensor was fabricated for DNA detection based on gold nanopartides (AuNPs) locked nucleic acid modified hairpin DNA probe (LNA-m-HpDNA) and enzymatic signal amplification. LNA-m-HpDNA dually labeled with 3'-thiol and 5'-biotin was efficiently assembled on the electrode via the interaction of thiol and gold. The stem-loop structure of LNA-m-HpDNA was unfolded after hybridization with target DNA, which forced biotin away from the electrode surface. Streptavidin-labeled horseradish peroxidase (Streptavidin-HRP) was subsequently immobilized on the electrode surface via the specific conjugation of biotin and streptavidin. Thereafter, the resulting HRP/target DNA-LNA-m-HpDNA (cDNA-LNA)/AuNPs modified Au electrode was successfully assembled for the construction of the DNA biosensor. Cyclic voltammetry and electrochemical impedance spectroscopy were carried out for the characterization of modified electrodes. Electrochemical reduction signal of benzoquinone was investigated in this work to indirectly analyze DNA hybridization via chronoamperometry. Under optimum conditions, the biosensor showed a good linear relationship between the current value and logarithm of the target DNA concentration ranging from 10 to 1000 pM with a low detection limit of 6.0 pM (S/N = 3). Furthermore, the DNA biosensor exhibited excellent discrimination ability to detect single-base mismatched DNA, three-base mismatched DNA and non-complementary DNA sequence. This strategy could provide a new platform for disease diagnose with high sensitivity. (C) 2012 Elsevier Ltd. All rights reserved.