Non-fried whole-wheat instant noodles feature high dietary fiber and balanced nutrition but suffer from poor rehydration, texture, and flavor. This study aims to improve the quality of these noodles through mixed fermentation of yeast and lactic acid bacteria (LAB). The rehydration characteristics, textural profile, sensory quality, microstructure, volatile flavor compounds, and in vitro digestibility of non-fried instant whole-wheat noodles were evaluated. Yeast primarily reduced rehydration time and improved mouthfeel, whereas LAB mainly contributed to the formation of a continuous and uniform gluten network as well as the enhancement of noodle flavor. Moderate addition of the mixed culture (1% yeast + 0.05% LAB) promoted the construction of a homogeneous gluten network in non-fried whole-wheat instant noodles, effectively reducing starch digestibility and estimated glycemic index (eGI). However, excessive addition caused opposite effects on these noodles. This study provides an effective processing strategy for the production of high-quality, low-eGI whole-wheat instant noodles, which are suitable for people pursuing healthy diets and controlling blood sugar levels.