Olivetol is an important polyphenol compound and intermediate in the synthesis of cannabinoids possessing many types of biological activities. A facile electrochemical sensor for olivetol was fabricated based on p-L-serine, and copper oxide (CuO) nanoparticles modified carbon paste electrode (p-L-serine/CuO/CPE). The proposed p-L-serine/CuO/CPE was applied to the electrochemical detection of olivetol by cyclic voltammetry (CV) and differential pulse voltammetric (DPV). Through the characterizations of materials and modified electrodes, the p-L-serine/CuO/CPE exhibited enhanced electrochemical signals for olivetol compared to bare CPE and CuO/CPE in both CV and DPV methods. Under the optimized conditions, the proposed p-L-serine/CuO/CPE showed a good quantitative analysis ability and a wide analysis range from 20 to 100 μmol L−1 of olivetol with a limit of detection of 1.04 μmol L−1. Based on the reproducibility, repeatability, and stability exhibited by this fabricated sensor and the cheap and accessible raw materials, the p-L-serine/CuO/CPE became a novel determination choice for olivetol in the electrochemical method with the advantages of being cost-effective and convenient.