Conventional reactive dyeing generates colored effluents with large amounts of salt and alkali. Herein, we firstly used sodium hydroxide to pretreat cotton fabric to obtain different wettability. Then the non-aqueous dyeing system was applied to the eco-friendly dyeing and washing process of cotton fiber. Meanwhile, Fourier Transform Infrared Spectrometer (FT-IR), Zeta potential, and X-ray Powder Diffraction (XRD) analysis had been performed to analyze the difference of cotton fibers before and after pretreatment. Furthermore, the effect of pretreatment on the adsorption behavior was investigated by molecular dynamics simulations (MD). As compared with untreated cotton, the about 23.0 percent increase in K/S value was achieved for azo reactive dyes. Findings from FT-IR, Zeta potential, and XRD spectrum showed that the number of available -OH of cotton involved in the dyeing increased after pretreatment. MD simulations demonstrated that the order of carbon atom on pretreated fibers and desorption coefficient of dyes decreased. While, the interaction energy between dye molecules and pretreated cotton fibers increased. As a result, reactive dye molecules showed a faster adsorption behavior on the pretreated fiber surface. Therefore, the pretreatment of cotton fabric is an effective way to improve the utilization rate of reactive dyes in non-aqueous media and realize the clean dyeing of reactive dyes.