The objective of this study is to determine the influence of operational parameters of CO2 Huff-n-Puff EOR process in the Eagle Ford shale oil reservoirs using response surface methodology (RSM). Single-factor analysis was first conducted for establishing the Box-Behnken model in RSM. We selected the primary depletion time, gas injection time, cycle number, production time per cycle, and injection rate as the primary input variables using RSM. The cumulative oil production and net present value are optimized as the output factors. After that, Design of Expert 12 software was used to design the experimental table for the above setting factors. Corresponding to the results taken from the optimization, the most significant factor is injection rate, followed by injection time, cycle number, and primary depletion time, and production time per cycle is the least significant. Additionally, the optimum responses were found as primary depletion time of 2.37 years, injection time of 3.4 months, cycle number of 3, production time per cycle of 2.2 years, and injection rate of 5000 MSCF/D. Moreover, correlation coefficient (R2) of the regression model is 0.9977, and adjusted model R2 is 0.8898, which further indicates that the model has good reliability. Results show that RSM is a useful technique for optimization, and it also provides insights into optimizing and designing the CO2 Huff-n-Puff process in the shale oil reservoirs.