The nickel aluminium layered double hydroxides(NiAl-LDH) were synthesized by coprecipitation method.With the layer by layer(LBL) self-assembly of polystyrene sulfonate sodium(PSS) and NiAl-LDH，the PSS/NiAl-LDH multilayer electrode was constructed，and applied in the determination of glucose.The X-ray diffraction，Infrared and SEM spectra of NiAl-LDH showed that it had the typical LDH characteristic peaks and morphology.The UV-Vis spectrum was used to monitor the uniform LBL self-assembly of PSS and NiAl-LDH.Electrochemical study showed that glucose could be effectively catalyzed oxidation with NiAl-LDH mutlitlayer electrode.Under the optimal conditions，the sensor showed a good linear relationship in the range of 5.0×10-7-6.6×10-4 mol/L for glucose with a sensitivity of 8.9×10-4A?L?mol-1 and a detection limit(S/N=3) of 2.8×10-7 mol/L.