Poly-diallyl-dimethyl-ammonium chloride (PDDA) solution was used to disperse carbon nanotubes (CNTs) to form a stable PDDA-CNTs aqueous dispersion. The negatively charged glucose oxidase (GOx) and positively charged PDDA-CNTs composite were used to prepare multilayer biosensing films on glassy carbon electrodes (GCE) via layer-by-layer (LBL) self-assembly technique. The optimum number of layers on GCE was 4. A mixture of 3-dimethyl (methacryloyloxyethyl) ammonium propane sulfonate (DMAPS) and Graphene (GR) was dropped on the multilayer films to prepare a bacteriostatic glucose biosensor. The results show that CNTs could evenly disperse in the PDDA films and the multilayer PDDA-CNTs films can significantly improve the catalytic current response toward glucose (Glu). The biosensor could detect glucose linearly from 16.5 to 214.3 mM. The bacteriostatic properties of the sensor were ensured by the bacteriostatic characteristic of DMAPS. (C) The Author(s) 2017. Published by ECS. All rights reserved.