An ultrasensitive DNA biosensor was constructed in this work by using graphene oxide nanosheets (GONs) as the multi-site platform for probe DNA immobilization. First, L-cysteine (L-cys) was self-assembled on a gold electrode surface based on the Au-S chemistry. Then the GONs and amino modified probe DNA was covalently grafted to L-cys/AuE surface in turn, with the aid of 1-(3-Dimethylanminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxy succinimide (NHS). The layer upon layer assembly process was characterized with atomic force microscopy and electrochemical methods. Due to the nano-size effect and multiple site characteristic of GONs, the immobilization density of the probe DNA was determined to be as high as 5.72 × 10⁻¹⁰ mol cm⁻² using methylene blue as the redox probe. The hybridization experiments showed that the biosensor can quantitatively detect target DNA in a wide range from 1.0 × 10⁻¹⁵ M to 1.0 × 10⁻⁹ M with a detection limit of 5.0 × 10⁻¹⁶ M. The selectivity experiments showed that the sensing system could accurately discriminate the complementary sequence from the base mismatched and non-complementary sequences.