In this study, we explored the technical parameters of tree peony seeds oil (TPSO) after their treatment with radio frequency (RF) at 0 °C–140 °C, and compared the results with microwave (MW) and roasted (RT) pretreatment in terms of their physicochemical properties, bioactivity (fatty acid tocopherols and phytosterols), volatile compounds and antioxidant activity of TPSO. RF (140 °C) pretreatment can effectively destroy the cell structure, substantially increasing oil yield by 15.23%. Tocopherols and phytosterols were enhanced in oil to 51.45 mg/kg and 341.35 mg/kg, respectively. In addition, antioxidant activities for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric-reducing antioxidant power (FRAP) were significantly improved by 33.26 μmol TE/100 g and 65.84 μmol TE/100 g, respectively (p < 0.05). The induction period (IP) value increased by 4.04 times. These results are similar to those of the MW pretreatment. The contents of aromatic compounds were significantly increased, resulting in improved flavors and aromas (roasted, nutty), by RF, MW and RT pretreatments. The three pretreatments significantly enhanced the antioxidant capacities and oxidative stabilities (p < 0.05). The current findings reveal RF to be a potential pretreatment for application in the industrial production of TPSO.