In this paper, a novel electrochemical method is proposed for the determination of 5-hydroxytryptophan (5-HTP) based on a carbon nanosheets (CNSs)-modified electrode. The CNSs were prepared by a one-step flowing electrolysis using a home-made device. The surface morphology and composition of the CNSs were characterized by scanning electron microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The cyclic voltammetry method was used to study the electrochemical behavior of 5-HTP on the CNSs-modified electrode. The results suggested that the modified electrode exhibited high electrocatalytic activity, and that the anodic peak current of 5-HTP was significantly enhanced on the modified electrode. A differential pulse voltammetry method was developed for the determination 5-HTP at the modified electrode. In the optimum experimental conditions, the linear calibration range varied from 5 × 10⁻⁸ mol L⁻¹ to 1 × 10⁻⁶ mol L⁻¹, with a detection limit of 3 × 10⁻⁸ mol L⁻¹. The proposed method was further successfully applied for the determination of 5-HTP in rats' brain tissue samples, with the recovery ranging from 90.3% to 98.0%.