Objective: In this study, ultra performance liquid chromatography-high resolution quadrupole time of flight mass spectrometer (UPLC-Q-TOF/MS) was established to determine β-sitosterol, brassicasterol, and campesterol in camellia oleifera seed oil. Methods: Using 6-ketocholestanol as the internal standard and performing sample extraction by heating it for 30 min and refluxing it with 50 mL ethanol. After centrifugation and layering, three sterols were separated by Xbridge BEH C₁₈ (100 mm × 4.6 mm, 2.5 μm) Chromatographic column elution. Finally, ionized by atmospheric pressure chemical ionization source and analyzed by positive ion mode. Results: The internal standard curves of the three sterols showed an observable linear relationship in the range of 0.1～10.0 mg/L, while the correlation coefficient R² was both greater than 0.999 2. The average recoveries at 0.5, 1.0, 2.0, and 10.0 times of background value were in the range of 84.5%～99.5%. The relative standard deviation (RSDs) was ranged from 3.92% to 7.44%. Moreover, the limits of detection were 1.0 mg/kg and the limits of quantitation were 3.0 mg/kg, respectively. Conclusion: The method was simple in operation, low in solvent consumption, and toxicity. It had good recoveries and reproducibility that could be applied for the accurate determination of three sterols in camellia oleifera seed oil.