Abstract: To reveal the differences in dyeing behavior of multi-component fibers in blended yarns, the dyeing mechanism of viscose fiber and viscose/polyester blended yarn (70/30, referring to a mass ratio of 70% viscose fiber and 30% polyester fiber) in the reactive dye X-3B system was systematically investigated, using stable isotope technology combined with conventional characterization methods. Results showed that as the dye concentration increased, the δD (hydrogen stable isotope value) in the viscose/polyester blended yarn exhibited a depletion phenomenon, while pure viscose fiber showed a slight enrichment, indicating significant differences in the dyeing behavior of the two fibers during the dyeing process. The variation range of δ¹⁸O (oxygen stable isotope value) was 1‰, indicating that reactive dyes have a certain impact on oxygen isotopes, independent of dye concentration. Temperature had a significant impact on the dyeing effect. As the temperature increased, the δ¹⁸O value rebounded, and more dye remained on the fiber surface, reducing deep binding. Low temperatures favored the stability of hydrogen bonds, enhancing the dye's adsorption capacity, while high temperatures increased fiber swelling, causing more dye to remain on the fiber surface and reducing deep binding, leading to a depletion trend in δD (decreasing from −60.4‰ to −63.9‰). Increasing the alkali concentration led to a depletion trend in δD, enhanced the binding ability of hydrogen bonds, and tightened the bond between dye and fiber, while the change in δ¹⁸O was more complex, influenced by various factors, and showed no obvious pattern. In all dyeing processes, δ¹³C (carbon stable isotope value) was hardly affected because C acts as the basic skeleton of the fiber. Therefore, reactive dyes significantly altered the hydrogen and oxygen stable isotope ratios of the fiber during the dyeing process, and were jointly regulated by dye concentration, temperature, and alkali concentration.