A method, based on the inhibition of an ultrathin polypyrrole-glucose oxidase (PPy-GOx) potentiometric biosensor response, is described for the detection of Cu2+, Hg2+, Cd2+ and Pb2+ ions. Based on experimental conditions (0.2 M pyrrole, 500 U mL-1 GOx, and an applied current density of 0.05 mA cm-2 and a polymerization period of 500 s) previously published by us, PPy-GOx films of approximately 55 nm thick were used to demonstrate the inhibitive potentiometric detection of selected trace metals down to 0.079 μM Cu2+, 0.025 μM Hg2+, 0.024 μM Pb2+ and 0.044 μM Cd2+. Furthermore, good linear concentration ranges were achieved for Cu2+ (0.079-16 μM), Hg2+ (0.025-5 μM), Pb2+ (0.10-15 μM) and Cd2+ (0.04-62 μM). The analysis of the nature of the inhibition of glucose oxidase in the PPy-GOx biosensor by these metals was achieved by Dixon and Cornish-Bowden plots. The shapes of the curves (exponential decay, parabolic and linear) obtained for the inhibitors suggest that the inhibition by the metal ions may not be exclusively directed at the essential -SH group, but involve additional binding sites of the enzyme. Dixon and Cornish-Bowden plots suggest that the inhibition is competitive for Cd2+, while non-competitive inhibition was observed for other metal ions. The ultra-thin PPy-GOx film enabled improved permeability to the metal inhibitors than possible with conventional biosensors with thicker films and, hence, better reflects the actual inhibition effect of the trace metals on the enzyme activity. The use of the ultra-thin film also eliminated the usual need for incubation of the enzyme electrode for a long period in the presence of the inhibitors. Furthermore, a rapid recovery of the enzyme activity was achieved by simply washing the electrode with water and storing in phosphate buffer for 10-15 min. The proposed biosensing approach was successfully used for the detection of individual trace metals in tap water, achieving a 98-101% recovery.