<p>In this article, chitosan/graphene oxide/glutathione nanofibers have been prepared as nano biosensors by electrospinning method. The surface morphology of nanofibers and the identification of functional groups have been investigated by field emission scanning electron microscopy (FESEM) and Fourier-transform infrared spectroscopy (FTIR) analysis. Then, according to the results of Autolab analysis test, 2.5 wt.% chitosan/graphene oxide sample was selected as optimal. In the last stage of presence of glutathione protein, two heavy metals lead and copper were detected in the water environment. The purpose of this article, seeks to design a nano biosensor for faster, more accurate and more cost-effective detection of heavy metals in the dissolved environment, which has the ability to be used in medical sciences, environmental food industries. The importance of this article is in expanding the use of nanoscale biosensors. These nano biosensors in this article are produced by electrospinning process. Chitosan was used as one of the biopolymers that has been widely used in sensor production.</p>