The study was conducted in the Laboratories of the College of Agriculture, University of Basrah, Iraq in 2023 to detect the bioactive compounds in hemp seed oil using GC-MS technology. The detection device by GC-MS showed 33 bioactive compounds in hemp seed oil. The results indicate that hemp seed oil contained 16 unsaturated fatty acids. It was found that the content of hemp seed oil was high in the two unsaturated fatty acids, Linoleic acid methyl ester and Oleic acid methyl ester. The peak area percentage of these two unsaturated fatty acids was about 44.15% and 37.45%, respectively. The two unsaturated fatty acids, Palmitic acid methyl ester, and Stearic acid methyl ester recorded a lower peak area percentage, reaching 10.05% and 5.98%, respectively. The other fatty acids detected in the hemp seed oil sample recorded very lower peak area percentage ranging within the range (0.003% - 0.730%). The unsaturated fatty acid α-Linolenic acid, methyl ester in hemp seed oil recorded the lowest peak area percentage of all the compounds detected by GC-MS, which was 0.003%. The data indicated the presence of six unsaturated fatty acids with high molecular weights, namely Erucic acid, 11-Eicosenoic acid, methyl ester, n-Pentacosanoic acid, n-Tricosanoic acid, n-Heneicosanoic acid, and Heptacosanoic acid. The results revealed that hemp seed oil contains Dioxaphetyl butyrate (0.03%), Naloxone (0.03%), Odorine (0.10%), and Pretazettine (0.01%) for medical and therapeutic uses. Hemp seed oil contains the two most important bioactive compounds of the phytosterols, namely Beta-Sitosterol and Campesterol (0.13% and 0.02%).