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A two-stage sequential pretreatment including caustic mercerization (CM) and liquid ammonia (LA) treatment was applied to investigate the influence on dyeing performance and handle of knit cotton fabric, and the relationship between dye size and dyeing properties. Various techniques were applied to characterize all the treated fabrics. X-ray diffraction (XRD) and Fourier-transform infrared (FTIR) analyses of the treated fabrics confirmed that both sequential treatments decreased the crystallinity of cotton fabric more than only the CM or LA treatment. The pattern of cellulose I was transferred to a mixed configuration of cellulose II and cellulose III after the CM/LA or LA/CM treatment. Thermal performances measured by thermogravimetric analysis (TGA) and differential thermogravimetry (DTG) techniques showed that the thermal stability of the treated cotton only marginally decreased. The wicking height increased after the sequential CM/LA treatment, indicating that the hydrophilicity of the fabric increased. The dye absorption and color uniformity were better for the reactive dye with a smaller molecular weight (Reactive Red 2) compared with the one with a larger molecular weight (Reactive Red 195). The total dye fixation efficiency (T%) increased to 72.93% and 73.24% for Reactive Red 2 dyeings of CM/LA- and LA/CM-cotton fabric from 46.75% of the untreated fabric, respectively; the T% increased to 65.33% and 72.27% for Reactive Red 195 dyeings of CM/LA- and LA/CM-cotton fabric from 35.17% of the untreated fabric, respectively. The colorfastness and dye exhaustion and fixation percentages of the samples were enhanced after the treatments. Furthermore, compared to the single CM or LA treatment, the softness handle properties were further improved after the fabrics were sequentially treated by CM/LA. The developed pre-treatment of CM/LA can be used in the textile industry to promote the dyeability, handle, and mechanical properties of knit cotton fabrics.