Whey protein was pretreated with multi-frequency ultrasound in mono, dual, and tri-frequency modes. The effect of multi-frequency ultrasound pretreatment on the hardness, chemical forces, and microstructure of acid-induced whey protein gel was investigated. Whey protein gels pretreated with dual and tri-frequency ultrasound showed higher hardness (p3.35 mm) in those samples were resisted to gastric digestion. Moreover, the tri-frequency ultrasound pretreatment of whey protein gel released the least free amino group during gastric digestion. In contrast, whey protein gel with the mono-frequency ultrasound pretreatment released the highest amount of free amino acid group during intestinal digestion. Findings from this study suggested that gel hardness and network density could modulate the digestion behaviors of protein gels.