The aim of this study is to develop an eco-friendly reduction process of indigo as an alternative choice. Hansenula misumaiensis was used and their reducing activity toward synthetic indigo as well as natural indigo was estimated by dyeing test in terms of indigo dye uptake. The changes in K/S value and pH were monitored on the time-based measurements. Also, reduction duration was evaluated. On the basis of the results described in this study, it was confirmed that Hansenula misumaiensis reduced indigo. Reducing power of Hansenula misumaiensis reached to maximum in two days. It can be possible to develop eco-friendly process of indigo reduction using Hansenula misumaiensis by the optimization of strain culture conditions and the optimization of reduction conditions.