CO2 microbubbles have recently been used in enhanced oil recovery for blocking the high permeability zone in heterogeneous reservoirs. Microbubbles are colloidal gas aphrons stabilized by thick shells of polymer and surfactant. The stability of CO2 microbubbles plays an important role in improving the performance of enhanced oil recovery. In this study, a new class of design of experiment (DOE)—definitive screening design (DSD) was employed to investigate the effect of five quantitative parameters: xanthan gum polymer concentration, sodium dodecyl sulfate surfactant concentration, salinity, stirring time, and stirring rate. This is a three-level design that required only 11 experimental runs. The results suggest that DSD successfully evaluated how various parameters contribute to CO2 microbubble stability. The definitive screening design revealed a polynomial regression model has ability to estimate the main effect factor, two-factor interactions and pure-quadratic effect of factors with high determination coefficients for its smaller number of experiments compared to traditional design of experiment approach. The experimental results showed that the stability depend primarily on xanthan gum polymer concentration. It was also found that the stability of CO2 microbubbles increases at a higher sodium dodecyl sulfate surfactant concentration and stirring rate, but decreases with increasing salinity. In addition, several interactions are presented to be significant including the polymer–salinity interaction, surfactant–salinity interaction and stirring rate–salinity interaction.