Due to its high amylopectin content and absence of gluten proteins, highland barley flour exhibits poor dough-forming capacity and processability. This study investigated combined modification strategies, Lactobacillus plantarum fermentation and pullulanase treatment, to improve the flour’s physicochemical and processing characteristics. Results indicated that these treatments significantly altered the morphological appearance and nutritional composition of highland barley flour, as well as increasing the amylose content. The group subjected to enzymatic hydrolysis followed by fermentation achieved the highest amylose content of 29.09%, while the amylopectin content decreased to 39.99%. Meanwhile, the content of soluble dietary fiber rose from 18.32% to 60.64%. Structural analysis revealed that enzymatic hydrolysis and fermentation could disrupt the hydrogen bond network and double-helix structure of starch, thereby reducing gelatinization parameters, with the most pronounced effect observed in the combined treatment of prior enzymatic hydrolysis followed by fermentation. For processing properties, this combination markedly improved the water-holding capacity and reduced the oil-holding capacity of the flour. Additionally, this combined modification exerted the most prominent enhancement on dough springiness, chewiness, viscoelasticity and rheological properties. Consequently, the combined modification of enzymatic hydrolysis and fermentation is a promising strategy to improve the nutritional value and processability of highland barley flour.