Indigo dyeing for cotton fibers can achieve ideal color depth in silicone non-aqueous medium dyeing system and solve some environmental pollution problems which were caused by the traditional water bath dyeing. However, the rubbing fastness of indigo-dyed cotton fiber is poor, which will influence the spinning process. In this investigation, three fixing agents commonly used in the market were screened to treat the indigo-dyed cotton fibers and their working mechanism was also studied. The results of GPC and FTIR showed that all the three fixing agents were polyurethanes which have a similar narrow molecular weight distribution and they could combine with indigo-dyed cotton fiber with intermolecular forces (hydrogen bonds) and ionic bonds with indigo-dyed cotton fiber. Compared with these fixing agents, fixing agent J has a better effect on reducing the friction coefficient of fiber because cotton fiber was the smoothest after fixing. After adding some cationic surfactant during fixing, the effect of fixation was influenced because there was a competition relation between the fixing agent and surfactant. From the spinning process, the rubbing fastness of fiber treated with fixing agent can meet the spinning requirements. 100 kg/d industrial application provides compelling proof of indigo dyeing in non-aqueous medium dyeing system, because this dyeing technology can change the complex process of traditional indigo dyeing, the monotonous style, and the serious waste water pollution for cowboy wear production.