Abstract: In order to study the effect of physical synergistic enzymatic treatment on the structural characteristics of highland barley starch, ultrasonic and pressure heat were combined with pullulanase to treat highland barley starch. The particle morphology, crystal structure, functional group structure and particle size of treated samples were determined. The results showed that the contents of resistant starch and amylose were increased while amylopectin was decreased. The original structure of starch granules of highland barley was seriously damaged by the modification, showing a clumped structure, rough surface, full of wrinkles, cracks and holes, and the polarized cross disappeared. It was also found that the average particles size of highland barley starch and the number of large particles increased. Meanwhile, the crystallinity of highland barley starch changed from A-type crystal to B-, C- and V-type which lead to the crystallinity increased. The modified treatment did not produce new chemical groups and chemical bonds, but changed the internal structure of barley starch rearrangement. In addition, compared to untreated, the order degree (DO) of the modified barley starch increased, and the value of DO treated with pullulanase was the largest. The modification treatment made the starch molecules into smaller particles then formed more denser and larger starch crystals.