An amperometric pyruvate-sensing electrode was prepared by immobilizing pyruvate oxidase (PyOx) on a polyion complex membrane. First, aqueous solutions of poly-L-lysine and poly(4-styrenesulfonate) were successively placed on a mercaptopropionic acid-modified gold surface and allowed to dry. A photo-crosslinked poly(vinyl alcohol) layer containing PyOx was then formed on the poly-L-lysine/poly(4-styrenesulfonate)-complex layer. The polyion complex layer was effective For eliminating electrochemical interferents such as L-ascorbic acid, uric acid, L-cysteine and acetaminophen, whereas the hydrogen peroxide produced through the PyOx-catalyzed reaction permeated easily through the layer. This resulted in a high sensitivity (detection limit, 50 nM) and a low interference level (e.g. the ratio of response for L-ascorbic acid to that for the same concentration of pyruvic acid, 0.18). The electrode could be used for determining phosphoric acid (detection limit, 0.2 mu M), since PyOx consumes phospholic acid as the co-substrate during the course of pyruvate oxidation. Further, an amperometric urea-sensing electrode (detection limit, 0.5 mu M) was prepared by coupling the phosphate-sensing system with urea amidolyase which catalyzes an ATP-consuming urea hydrolyzation.