Aflatoxin M-1 (AFM(1)) and Ochratoxin A (OTA) are a class of mycotoxins produced as secondary metabolites mainly by Aspergillus species which contaminate a large variety of food and feed commodities. These compounds elicit a wide spectrum of toxicological and carcinogenic effects, affecting human and animal health. Consequently, there has been an increasing need to establish simple and sensitive methods for their detection. Among different approaches used for analysis of toxins, electrochemical detection seems especially promising due to high sensitivity, feasibility of low cost, compatibility with portability and miniaturization. Herein, a strategy based on a competitive immunoassay that uses a secondary antibody conjugated with an enzyme (alkaline phosphatase) as a tag was explored for the voltammetric detection of mycotoxins (OTA and AFM(1)) using modified gold screen printed electrodes (AuSPE). The presented biosensor was challenged in red wine and milk samples with no need for pre-treatment or pre-concentration of the sample extract. The analytical signal was proportional to the toxin concentration in a wide working linear range, showing an excellent limit of the detection at ng mL(-1). Additionally, AuSPE modified with self-assembled monolayers based on different types of alkanethiols (long and short chains) were tested and compared in terms of electron transfer resistance. Combining all most desirable aspects of a good biosensor such as high sensitivity, low costs (use of inexpensive and disposable SPE and the reagents volume reduction to the size of a droplet), short analysis time and simple but effective cleaning-up technique, we report in this manuscript a very promising tool for widespread biosensing applications. (C) 2017 Elsevier Ltd. All rights reserved.