The present work describes the construction and application of a new DNA biosensor for detection of aflatoxin M1. In order to immobilize a thiol-modified single stranded DNA (ss-HSDNA) probe that specifically bound aflatoxin M1, a self-assembled monolayer of cysteamine and gold nanoparticles on the SAM were prepared on gold electrodes, layer-by-layer. The assembly processes of cysteamine, gold nanoparticles, and ss-HSDNA were monitored with the help of electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques. K-3[Fe(CN)(6)]/K-4[Fe(CN)(6)] solution was used as a redox probe for electrochemical measurements. The biosensor provided a linear response to aflatoxin M1 over the concentration range of 1-14 ng/mL with a standard deviation of +/- 0.36 ng/mL Finally, the biosensor was applied to a series of real milk samples. (C) 2011 Elsevier B.V. All rights reserved.