The volatile flavor compounds of ghee were isolated using sub-critical CO2 extraction. The effect of extraction pressure (3 to 8 MPa), temperature (3 to 38 °C), and time (19 to 221 min) on the extractability of flavor compounds i.e., δ-dodecalctone (C12), δ-tetradecalctone (C14), 3-ethyl-3-methyl heptane and total extraction yield as response variables were studied. The experiments were conducted using a central composite rotatable design of the independent variables. The study revealed that the pressure had a positive effect on the extractability of all three compounds. The optimized sub-critical CO2 extraction pressure, temperature and time for maximum recovery of these compounds were observed at 7 MPa, 10 °C and 66 min, respectively. The values of yields of δ-dodecalctone (C12), δ-tetradecalctone (C14), 3-ethyl-3-methyl heptane, and total extraction yield at the optimized conditions were found to be were found to be 216.54, 109.7, 64.82 ng .g−1 and 5.89% (w/w), respectively.