Cotton fabrics are prone to wrinkles and can be treated with citric acid (CA) to obtain good anti-wrinkle properties. However, the yellowing of the CA-treated fabrics is one big obstacle to the practical application of citric acid. The changing sequence order of CA anhydride and unsaturated acid (the reason for yellowing), such as aconitic acid (AA), has not been investigated. Herein, Fourier transform infrared (FTIR) spectroscopy, two-dimensional correlation spectroscopy (2Dcos), and Gaussian calculation were employed to characterize the reaction mechanism between CA with cellulose. FTIR spectra of the CA-treated fabrics heated under different temperatures were collected and further analyzed with 2Dcos. The results indicated the changing sequence order: 1656 cm⁻¹→1784 cm⁻¹→1701 cm⁻¹, (“→” means earlier than), i.e., unsaturated acid→anhydride→ester. Moreover, a change of Gibbs free energy (ΔG) showed that trans-AA (ΔG = −22.10 kJ/mol) is more thermodynamically favorable to be formed than CA anhydride 1 (ΔG = −0.90 kJ/mol), which was proved by Gaussian computational modeling. By taking cellobiose as a model of cellulose, the ΔG results proved that O(6)–H(6) on the glucose ring is the most likely hydroxyl to react with anhydride originated from CA or AA, especially with the terminal carbonyl group.