CO2-responsive fluid (CRF) is a surfactant solution characterized by low viscosity during the injection stage, and by a viscosity increase after reaction with CO2, which achieves mitigation of CO2 channeling. CRF has been applied in several pilot tests for CO2 conformance control in the Xinjiang Oilfield, China. However, its thickening characteristics (i.e., the viscosity change behavior) are not clearly defined, and the mechanism of conformance control requires further understanding. In this study, the static thickening behavior and reservoir adaptability (e.g., reservoir temperature and formation water salinity) of CRF were investigated through viscosity evaluation and microscopic characterization methods. Combining core displacement experiments and nuclear magnetic resonance (NMR) analysis, the CO2 conformance improvement of CRF in heterogeneous cores was examined. Results indicated that the viscosity of CRF after reacting with CO2 increased with the concentrations of reagent A (tertiary-amine single-chain surfactants) and reagent B (quaternary ammonium gemini surfactants), reaching a maximum of 460 mPa·s with the optimal composition of 1.5% reagent A and 0.2% reagent B. A mathematical equation for static thickening behavior was established to predict viscosity variation, providing a basis for rapid screening before oilfield applications. FTIR analysis confirmed that tertiary amine groups in the CRF molecular structure possessing CO2-responsive functionality, as thickening occurred through protonation reaction. SEM observation showed that CRF exhibited a lamellar morphology before reaction with CO2, while three-dimensional (3D) network structures formed after reaction due to micelle formation. NMR T2 spectra demonstrated that CRF had significant plugging and CO2 conformance control effects in the three-layer heterogeneous core with the permeabilities of 100/300/1500 × 10−3 μm2, effectively delaying CO2 channeling. This study provided CO2-responsive thickening systems and technical guidance for further development and field-scale application of CRF for conformance control during CO2-EOR processes.