One of the most common mycotoxin contaminating feed and foodstuffs is Ochratoxin A (OTA). OTA has a chronic toxic effect and proved to be mutagenic, nephrotoxic, teratogenic, immunosuppressive, and carcinogenic. Aptamer with their specific affinity for OTA was used in this paper to create an analytical technique. Several methods have been reported for the determination of OTA in foods. However, most of these methods could not be applied to a complex food as green coffee because the interfering native fluorescent products made the quantification very difficult. In this work, we mixed two separations based techniques to identify and quantify OTA in green coffee. Aptamer assisted ultrafiltration as separation technique based on the size of molecules was applied to separate the free OTA, the quantification of OTA was established by a high-performance liquid chromatography (HPLC-FD) with a limit of detection (LOD) of 0.05¿ng/mL for OTA. Artificially contaminated green coffee displayed a good range of OTA recoveries up to 97.7%. This method can be applied to the quantitative determination of OTA in green coffee at levels below the maximum levels proposed by the European Commission for green coffee. It also confirm that aptamers can be used as biorecognition element in diagnostic assays with commercial application for mycotoxin analysis.