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We present ion-sensitive conductometric glucose sensors prepared by layer-by-layer (LbL) nano self-assembly of single-walled carbon nanotube (SWNT) and enzyme glucose oxidase (GOx). The carboxylated SWNT and GOx are self-assembled alternatively with a positively charged polyelectrolyte, poly(diallyldimethylammonium chloride, PDDA). Quartz crystal microbalance (QCM) study and Fourier transform infrared (FTIR) spectroscopy demonstrate GOx is negatively charged, and it is possible to successively construct multilayer alternatively with PDDA. SWNT multilayer shows pH-dependent conductance at different pH buffer solutions, which decreases exponentially with an increase in pH. The concentration of glucose is electronically characterized based on the fact that hydrogen ions from glucose oxidation with the aid of GOx change the local pH value in the vicinity of SWNT multilayer thin-film, thereby yielding higher conductance. The overall sensitivity is 10.8 muA/mM and the resolution of device fabricated is 1 pM at the bias voltage of 0.6 V. The LbL assembled SWNT multilayer is proven to be versatile for sensing biochemical reactions with many other enzyme systems.