Abstract: Highland barley protein with weak gelation properties constrained its application. To explore the formation condition of highland barley protein gelation, highland barley protein was extracted by isoelectric precipitation method, and the effect of protein concentration, pH, heating temperature, heating time, urea, neutral salt and propanediol concentration on gelation properties were studied by texture analyzer and rheometer. Results indicated that gel started to form when the concentration was beyond 14% at alkaline condition (pH≥8) . The hardness of gel reached maximum when the heating temperature was 95℃ and heating time was 40min. With the increase of NaCl and propanediol concentrations, the hardness and storage modulus of the protein gel firstly increased and then decreased. However, the hardness and storage modulus significantly increased with the increase of urea concentration and the gel hardness was 90.29g when urea concentration was 7mol/L. Results showed that denaturation by urea destroyed hydrogen bonds between intramolecular and intermolecular chains and hydrophobic interaction with the result of protein unfolding and active groups exposing, which was in favor of gel formation.