D5, as an eco-friendly dyeing media instead of water, has also showed a successful development in the application of ecological dyeing and finishing. In this paper, molecular dynamics (MD) simulation methods were performed to further analyze the effects of different substituents on dyeing properties of disperse dyes based on a molecular perspective. The effect of substituents on solubility of disperse dyes was analyzed macroscopically by solvent accessible surface area (SASA), mean squared displacement (MSD), and diffusion coefficient (D). To explain the difference of dyeing uptake of disperse dyes with different substituents, interaction energy between dye, D5, and fiber polymers, and adsorption behavior were stimulated and calculated. The obtained results showed that both SASA and interaction energy between dye and D5 molecules decreased gradually with the substituent changing from N-ethyl to N-acetoxyethyl and N-cyanoethyl group. It led to the reduction of solubility in D5 media. The calculated adsorption speed and interaction energy between D5 and fiber molecules increased with the substituents ranging from N-ethyl to N-cyanoethyl and N-acetoxyethyl group. Accordingly, the D value decreased gradually. As a result, the dyeing uptake of disperse dyes increased from 85.3 to 93.3% and 95.5%, respectively. The results indicated that the key to improve dyeing uptake is to use substituents to regulate the supramolecular interaction between dye, D5, and fiber molecules.