In the past few decades, the Cannabis sativa L. hemp variety has been unfairly neglected because of its similarity to the illegal kind Cannabis indica L. used as a narcotic. The objective of this study was to evaluate the process of oil extraction from Cannabis sativa L. seeds by cold pressing, followed by extraction with supercritical CO2. Extraction optimization of hemp oil production was conducted using response surface methodology (RSM). The effects of temperature, temperature head presses, frequency, and nozzle size on oil recovery and quality parameters in the pressing experiments and the effects of extraction pressure, temperature and solvent flow rate in supercritical CO2 extraction experiments were investigated. In obtained hemp oils the following parameters were analysed: peroxide value, free fatty acids, insoluble impurities, moisture content, iodine value, saponification value, Anisidine value and Totox value. The composition of fatty acids, tocopherols, chlorophyll and carotene content was also determined. The influence of different natural antioxidants and their concentration on the oxidative stability of hemp seed oil was investigated. The results show that the oil recovery and quality was statistically significantly (ANOVA) affected by applied operational extraction process parameters. The residual oil in the pressed cake was extracted almost completely by supercritical CO2 in a newly designed supercritical fluid extraction system. By selecting the relevant process conditions of supercritical extraction, as well as by fractionation, it is possible to obtain hemp oil with different mass concentrations oil fatty acids, tocopherols and pigments. The essential oil of oregano in concentration of 0.05 % was the most efficient in protecting the hemp oil against oxidative deterioration. Because of the oil’s high quality, as well as the high content of high-quality components in hemp seeds which positively affect human health, this study is very significant regarding the small number of published studies on hemp seed oil.