Herein, carboxypeptidase Y (CPY) and thermolysin (TLN) were compared as sensing elements to develop an original biosensor for the direct detection of ochratoxin A (OTA) in olive oil. Electrochemical detection of OTA was performed using biosensors containing either CPY or TLN immobilized through glutaraldehyde vapor cross-linking onto gold interdigitated microelectrodes. Different parameters affecting biosensor sensitivity towards OTA were first optimized. TLN and CPY exhibited an optimal activity under the same conditions: 35 min cross-linking time, working pH of 7 and temperature of 25 degrees C. TLN and CPY biosensors exhibited comparable analytical performances. The conductometric response of both modified electrodes was linear up to 75 mu M. The limits of detection were also very similar (1 mu M for the CPY biosensor and 0.7 mu M for the TLN biosensor). For both enzymes, initial rate vs. OTA concentration plots could be described by hyperbolic curves consistent with the Michaelis-Menten model. K-M(app) and V-max(app), deduced from these curves, were 26 mu M and 3.3 mu S min(-1) respectively for the TLN biosensor. The biosensors signal was reproducible, relative standard deviations calculated from three consecutive measurements of standards in the 1-75 mu M range, being lower than 5%. The signals were also very stable over a 30 days period when biosensors were stored at 4 degrees C in 20 mM phosphate buffer between two measurements. The TLN biosensor was further evaluated for OTA determination in spiked olive oil samples. Recovery values were close to 100% demonstrating the suitability of this method for OTA screening in olive oil. Interestingly, OTA concentrations could be determined without pretreatment of the sample and no matrix effect was observed.