Generally, analytical Chemiluminescence (CL) technique is based on the measurement of the electromagnetic radiation (usually visible/near-infrared radiation) emitted by a chemical reaction [1]. On the other hand, in recent years, nanomaterials with the unique physical and chemical properties can amplify the CL signal and improved the sensitivity the various CL detection systems [2]. In this work, we synthesized graphene oxide (GO) by using improved hummer method. Then, the structural study of GO was performed by ultraviolet and visible (UV-Vis), spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and spectroscopy, fourier transform infrared (FTIR). This study revealed that GO were well separated from each other and dispersed in solution. These GO were used for enhancing of luminol– O2 CL reaction. Then, we found that paracetamol were capable of altering CL emission of GO enhanced luminol –O2. Based on this finding, a new and sensitive CL method for determination of paracetamol drug was developed. The effect of various chemical parameters on CL intensity were investigated and optimized. Under the optimum conditions, a linear relationship was obtained between the enhanced CL intensity and the concentration of paracetamol in the range of 7×10-6–5×10-5 M with a detection limit of 8 × 10-7 M. The relative standard deviation for 5 determinations of 2×10-5 M paracetamol was 3.39%. The method was successfully applied to the determination of paracetamol in pharmaceutical formulations.