A multifunctional bio-sensing chip was designed based on the electrochemiluminescent (ECL) detection of enzymatically produced hydrogen peroxide. Six different oxidases specific for choline, glucose, glutamate, lactate, lysine and urate were non-covalently immobilised on imidodiacetic acid chelating beads (glucose oxidase only) or on diethylaminoethyl (DEAE) anion exchanger beads, and spotted on the surface of a glassy carbon foil (25 mm(2) square), entrapped in PVA-SbQ photopolymer. The chip measurement was achieved by applying during 3 min a +850 mV potential between the glassy carbon electrode and a platinum pseudo-reference, while capturing a numeric image of the multifunctional bio-sensing chip with a CCD camera. The use of luminol supporting beads (DEAE-Sepharose) included in the sensing layer was shown to enable the achievement of spatially well defined signals, and to solve the hydrogen peroxide parasite signal which appeared between contiguous spots using luminol free in solution. The detection limits of the different biosensor were found to be 1 microM for glutamate, lysine and uric acid, 20 microM for glucose and 2 microM for choline and lactate. The detection ranges were 1-25 microM (uric acid), 1 microM-0.5 mM (glutamate and lysine), 20 microM-2 mM (glucose) and 2 microM-0.2 mM (choline and lactate). The ECL chip was used for the detection of glucose, lactate and uric acid in human serum matrix. Good correlations between measured and expected values were found without the need of internal calibration of the sample, demonstrating the potentiality of the ECL multifunctional bio-sensing chip.