A stem cell chip with peptide nanopatterned layer was fabricated to detect the effects of environmental toxins on human neural stem cells (HB1 x F3) electrochemically. The cell chip was recently developed as in vitro monitoring tool for determining the cell viability simply and rapidly compared to the conventional methods. However, cell chip composed of neural stem cells have not been reported due to the difficulties for maintaining its stemness and cell attachment on the artificial electrode surface, which is critical for sensitive detection of cell viability electrochemically. In this study, we fabricated peptide nanopatterned layer on gold electrode for increasing the affinity between the stem cell and an artificial electrode surface by self-assembly technique. After the confirmation of fabricated nanopatterned surface, neural stem cells were immobilized on chip surface and the viability was measured by electrochemical method. Thereafter, neural stem cells were treated with two kinds of common environmental toxins, and the intensities of reduction peak obtained by cyclic voltammetry (CV) were decreased with the increase of concentrations of environmental toxins. These electrochemical results were validated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Our newly developed stem cell chip can be used as useful label-free analysis tool for detecting drug effects or for assessing the toxicity electrochemically.