We report a novel enzyme-based microcantilever sensor by using layer-by-layer nanoassembly modification. A glucose oxidase (GOx) functionalized microcantilever underwent bending when it was exposed to glucose solutions. The magnitudes of bending were proportional to the concentrations of glucose. The cantilever bending was specific toward glucose, but not to other sugars such as mannose, fructose, or galactose. The flow rate effect on the cantilever bending response is also discussed. The bending mechanism was investigated, and the kinetic and thermodynamic analysis and experimental results showed that the conformational change of GOx and gluconic formation were the origin of cantilever deflection.