Supercritical fluid extraction with carbon dioxide (SC-CO2 extraction) was performed to isolate essential oils from the rhizomes of Cyperus rotundus Linn. Effects of temperature, pressure, extraction time, and CO2 flow rate on the yield of essential oils were investigated by response surface methodology (RSM). The oil yield was represented by a second-order polynomial model using central composite rotatable design (CCRD). The oil yield increased significantly with pressure (p < 0.0001) and CO2 flow rate (p < 0.01). The maximum oil yield from the response surface equation was predicted to be 1.82% using an extraction temperature of 37.6 °C, pressure of 294.4 bar, extraction time of 119.8 min, and CO2 flow rate of 20.9 L/h.