A novel sandwich-type noncompetitive electrochemical immunosensor was successfully developed for detecting diethylstilbestrol (DES). First, a novel label (EV/PtNPs-DES Ab) was fabricated by immobilizing numerous platinum nanoparticles (PtNPs) and antibody of DES (DES Ab) on the EnVision (TM) polymerase chelate (EV). Because DES has two symmetrical phenol groups in the formula, beta-cyclodextrin (beta-CD) modified on the glassy carbon electrode (GCE) was used to be combined with one phenol group of DES, then forming a host-guest inclusion complex on GCE. Since other structure analogues of DES which has phenol group in food samples could also react with beta-CD, thus the signal tag (EV/PtNPs-DES Ab) with DES's antibodies was used to further conjugated with another phenol group of DES to form a sandwich-type immunocomplex (beta-CD/DES/EV/PtNPs-DES Ab) on the sensor. This immunocomplex can trigger significant electro-catalytic current derived from the reduction of H2O2 for quantification of DES. Through two-step recognition of DES by beta-cyclodextrin and antibody in labels, a sandwich-type noncompetitive and signal-on immunoassay for DES was achieved. Moreover, the larger amount of PtNPs coupled with HRP on the label could significantly amplify the current and enhance sensitivity. Based on the optimal conditions, this sensor had a good linearity range of 1.00-100 pg mL(-1) and a low detection limit of 0.30 pg mL(-1) for DES (S/N = 3). The assay was employed for detecting trace DES in milk samples. The results were consistent with that of conventional HPLC method. This implies that this non-competitive immunoassay has potential applications in detection of other small organic molecules in food safety. (C) 2014 Elsevier B.V. All rights reserved.