In this work, the effect of microwave (MW), ε‐poly‐L‐lysine (ε‐PL), calcium propionate (CP), and their combinations on the shelf life and quality characteristics of highland barley fresh noodle (HBFN) was studied. Firstly, the mixed flour was treated by MW (800 W) for different time. Subsequently, HBFN was prepared by mixing flour and sterilized water with addition of 0.04% ε‐PL combined with different concentrations of CP (0.025%, 0.020%, 0.015%) and stored at 25 ± 1°C. Changes of total plate count (TPC), moisture content, water state, textural properties, color, and pH in HBFN were monitored during storage. The results indicated that microwave treatment (40 s) provided 30% reduction of initial TPC of the flour without decline of noodle quality. Shelf life of HBFN treated with MW + ε‐PL (0.04%) + CP (0.02%/0.025%) reached 80 and 88 hr, respectively, and had good edible qualities during storage.