Abstract: Hemp oil contains cannabidiol acid(CBDA), cannabidiol(CBD), cannabinol(CBN), Δ9-tetrahydrocannabi-nol(THC), tetrahydrocannabinol acid(THCA) and other cannabidiol substances. The properties of the matrix were fully considered, and a simple and rapid sample preparation method for extracting five cannabinoids from hemp oil was developed. A HPLC method for simultaneous detection of five cannabinoids in hemp oil was established by optimizing the chromatographic conditions. The five cannabinoids and other substances in the matrix were successfully separated based on this method. The method was verified by precision experiments, repeatability experiments, and recovery experiments. The results showed that the five cannabinoids had a good linear relationship in the range of 0.5~50 μg/mL, and the limits of quantification(LOQ, S/N=10) were between 0.33~0.80 mg/kg, the recovery rate was 77.1%~103.3% with RSD≤4.7%. A few cannabinoids were found in actual hemp oil samples from 4 domestic production areas in China. THC contents in these four samples were less than 0.3%, but the THC content of some products was higher than the upper limit in EU countries, and might cause some kinds of safety risk. The method developed in this article would lay a methodological foundation for the establishment of relevant determination method standards.