A new chemiluminescence-based one-shot biosensor for lactate is described. The lactate recognition system is based on lactate oxidase (LOx) and the transduction system consists of luminol, peroxidase from Arthromyces ramosus (ARP) and metallic aluminum, all immobilized in a polyion complex membrane. The measurement of the chemiluminescence in a luminometer when 1 mL of sample is injected into a conventional cell containing the disposable sensing membrane makes it possible to determine lactate. The compositions of the membrane and reaction conditions have been optimized to obtain adequate sensitivity. The one-shot biosensor responds to lactate rapidly, with the typical CL acquisition time being 2 min, with a linearized logarithmic dependence whose dynamic range was from 5 x 10(-5) to 4 x 10(-3) with a detection limit of 9.2 x 10(-6)M and a sensor-to-sensor reproducibility (relative standard deviation, R.S.D.) of 5.5% at the medium level of the range. The performance of the chemiluminescent one-shot biosensor was tested for the analysis of lactate in yoghurt, validating the results against an enzymatic reference procedure. The proposed method is quick, inexpensive and sensitive and uses conventional CL instrumentation.