Immunoassays are fast and sensitive techniques for analyte quantification, and their use in point-of-care devices for medical, environmental, and food safety applications has potential benefits of cost, portability, and multiplexing. However, immunoassays are often affected by matrix interference effects, requiring the use of complex laboratory extraction and concentration procedures in order to achieve the required sensitivity. In this paper we propose an integrated microfluidic device for the simultaneous matrix clean-up, concentration and detection. This device consists of two modules in series, the first performing an aqueous two-phase extraction (ATPE) for matrix extraction and analyte pre-concentration, and the second an immunoassay for quantification. The model analyte was the mycotoxin ochratoxin A (OTA) in a wine matrix. Using this strategy, a limit of detection (LoD) of 0.26 ng mL(-1) was obtained for red wine spiked with OTA, well below the regulatory limit for OTA in wines of 2 ng mL(-1) set by the European Union. Furthermore, the linear response on the logarithmic concentration scale was observed to span 3 orders of magnitude (0.1-100 ng mL(-1)). These results are comparable to those obtained for the quantification of OTA in plain buffer without an integrated ATPE (LoD = 0.15 ng mL(-1)). The proposed method was also found to provide similar results for markedly different matrices, such as red and white wines. This novel approach based on aqueous two-phase systems can help the development of point-of-care devices that can directly deal with real samples in complex matrices without the need for extra extraction processes and equipment.