Abstract: Highland barley were treated with three enzyme deactivation ways including steaming, frying and microwave baking, and no enzyme deactivation as the reference.Effect of different enzyme deactivation treatment on highland barley nutritional value and protein functional properties was researched.Results showed that highland barley protein and starch after steaming were respectively decreased by 29% and 23%, and protein decreased by26% after frying, while less effect on the basic components of highland barley after microwave baking. After steaming and frying, highland barley protein solubility, foaming capacity and foaming stability, emulsifying capacity and emulsifying stability decreased, water holding capacity of protein reduced after steaming, while it increased after frying.After microwave, solubility, water holding capacity, foaming capacity and foaming stability of highland barley protein remained respectively stable, emulsifying capacity and emulsifying stability increased.After enzyme deactivation, protein gel hardness of highland barley increased, and it reached maximum after microwave baking, higher than 18% compared with control. It indicated that microwave baking could maintain nutritional value and good functional properties of protein on highland barley, and microwave baking was the best way of enzyme deactivation, it was suitable for milling and food process of highland barley.