A novel strategy for the fabrication of an electrochemical label-free aptasensor for small-size molecules is proposed and demonstrated as an aptasensor for ochratoxin A (OTA). A long spacer chain of polyethylene glycol (PEG) was immobilized on a boron-doped diamond (BDD) microcell via electrochemical oxidation of its terminal amino groups. The amino-aptamer was then covalently linked to the carboxyl end of the immobilized PEG as a two-piece macromolecule, autoassembled at the BDD surface, forming a dense layer. Due to a change in conformation of the aptamer on the target analyte binding, a decrease of the electron transfer rate of the redox [Fe(CN)(6)](4-/3-) probe was observed. To quantify the amount of OTA, the decrease of the square wave voltammetry (SWV) peak maximum of this probe was monitored. The plot of the peak maximum against the logarithm of OTA concentration was linear along the range from 0.01 to 13.2 ng/L, with a detection limit of 0.01 ng/L. This concept was validated on spiked real samples of rice