Chinese quince seed (CQS) is an underutilized oil source and a potential source of unsaturated fatty acids and α-tocopherol-rich oil. Subcritical fluid (SCF) extraction is executed at lower pressures and temperatures than the pressures and temperatures used in supercritical fluid extraction. However, no studies on the SCF extraction of CQS oil are reported. Therefore, the objective of this study was to evaluate the use of SCF for the extraction of CQS oil and to compare the use of SCF with the classical Soxhlet (CS) and supercritical CO2 (SC-CO2) extraction methods. Response surface methodology (RSM) was used to investigate the extraction conditions: temperature (45–65 °C), time (30–50 min), and solvent/solid ratio (5–15 mL/g). The optimization results showed that the highest yield (27.78%) was obtained at 56.18 °C, 40.20 min, and 12.57 mL/g. The oil extracted by SCF had a higher unsaturated fatty acid content (86.37%–86.75%), higher α-tocopherol content (576.0–847.6 mg/kg), lower acid value (3.97 mg/g), and lower peroxide value (0.02 meq O2/kg) than extractions using CS and SC-CO2 methods. The SCF-defatted meal of oilseed exhibited the highest nitrogen solubility index (49.64%) and protein dispersibility index (50.80%), demonstrating that SCF extraction was a promising and efficient technique as an alternative to CS and SC-CO2 methods, as very mild operating conditions and an eco-friendly solvent can be used in the process with maximum preservation of the quality of the meal.