Traditional dyeing of cotton fabrics with reactive dyes has produced many environmental problems. Therefore, it is important to investigate the relationship between dye molecular structures and dyeing properties for cleaner procedures. Here, the properties of five azo-based reactive dyes with different substituents and reactive groups were studied. The dyes had similar molecular shapes, sizes, and chromophore structures. Differences were in the aromatic rings, the number of sulfonate groups, and the position of vinyl sulfonate on the benzene ring. The dye substantivity (S%), exhaustion (E%), reactivity (R%), fixation (F%), color strength, salt effects, alkali effects, and color fastness were investigated and analyzed. Different substituents on the dyes had a little effect on the ultraviolet-visible absorption maximum wavelength. In addition, the requirements of salt and alkali to produce the maximum color strength varied for different dye structures. The dye containing a naphthalene ring, three sulfonate groups, and a vinyl sulfonate at the para-position of the benzene ring produced the highest color strength and fixation values. Color fastness was almost the same for each dyed sample, except for the wet-rubbing fastness. The results should be useful for developing new dyes and environmentally friendly dyeing techniques.