A sensitive and reliable inhibitive amperometric glucose biosensor is described for rapid trace metal determination. The biosensor utilises a conductive ultrathin (55 nm thick) polypyrrole (PPy) film for entrapment of glucose oxidase (GOx) to permit rapid inhibition of GOx activity in the ultrathin film upon exposure to trace metals, resulting in reduced glucose amperometric response. The biosensor demonstrates a relatively fast response time of 20 s and does not require incubation. Furthermore, a complete recovery of GOx activity in the ultrathin PPy-GOx biosensor is quickly achieved by washing in 2 mM EDTA for only 10 s. The minimum detectable concentrations achieved with the biosensor for Hg2+, Cu2+, Pb2+ and Cd2+ by inhibitive amperometric detection are 0.48, 1.5, 1.6 and 4.0 μM, respectively. Also, suitable linear concentration ranges were achieved from 0.48-3.3 μM for Hg2+, 1.5-10 μM for Cu2+, 1.6-7.7 μM for Pb2+ and 4-26 μM for Cd2+. The use of Dixon and Cornish-Bowden plots revealed that the suppressive effects observed with Hg2+ and Cu2+ were via non-competitive inhibition, while those of Pb2+ and Cd2+ were due to mixed and competitive inhibition. The stronger inhibition exhibited by the trace metals on GOx activity in the ultrathin PPy-GOx film was also confirmed by the low inhibition constant obtained from this analysis. The biosensor was successfully applied to the determination of trace metals in tap water samples.