A novel cheap and simple amperometric glucose biosensor, based on the electrode modified with the Ni/Al layered double hydroxide (LDH) nanoflakes and chitosan (CHT), without glucose oxidase, is presented. The glucose biosensor based on monodispersed high active Ni/Al-LDH nanoflakes and CHT exhibits an appropriate linear range of 0.01-10mM and good operational stability. The amperometric sensor shows a rapid response at the potential value 0.48V. In addition, optimization of the biosensor construction, the effects of the applied potential, the scan rate as well as common interfering compounds on the amperometric response and human serum samples analysis of the sensor were investigated and discussed.