Immunoassays have a broad application range, from environmental and food toxicology to biomedical analysis, providing rapid and simple methods for analyte quantification. Immunoassays, however, are often challenging at nM and sub nM concentrations and are affected by detrimental matrix interference effects, as is the case of the detection of ochratoxin A (OTA) and Aflatoxin B1 (AFB1). These are widespread mycotoxins found in food and feed, with serious potential implications for human health. This work demonstrates the use of polymer-salt aqueous two phase systems (ATPSs) for the simultaneous concentration of mycotoxins and neutralization of matrix interference. In particular, polyethylene glycol (PEG)-phosphate salt ATPSs were used to enhance the detection sensitivity of OTA and AFB1 in wines and beer by an indirect competitive ELISA. Using this methodology it was possible to quantify both analytes spiked in red wine with limits-of-detection (LoD) down to 0.19ng/mL and 0.035ng/mL, respectively, with results comparable to those obtained using solutions of toxins in phosphate buffered saline (PBS) buffer (0.7ng/mL and 0.009ng/mL, respectively). Furthermore, a very low matrix-to matrix variability was observed, with LoD and half inhibitory concentration (IC50) values of 5.17±1.08 and 33.2±3.5ng/mL (±SD) obtained in the detection of OTA spiked in red and white wines, beer or PBS buffer. These results indicate the potential of ATPS as a fast and simple concentration step and in providing matrix-independent analyte quantification for enhanced immunoassay sensitivity below regulatory levels.