In this study, the effect of alginate and/or transglutaminase (TGase) in combination with ultrasonic treatment on the heat-induced gel quality, water-holding capacity (WHC) and myosin conformational changes of unwashed surimi was investigated. The results showed that the addition of trehalose (4.5%, m/m) and/or TGase (0.5%, m/m) together with ultrasonic treatment increased the gel strength by 35.11%−169.25%. Trehalose prevented protein unfolding while TGase had the opposite effect. However, ultrasonic treatment inhibited the catalytic activity of TGase, thereby reducing the gel strength to 4 737.23 g·mm, decreasing the relative content of free water to 19.52%, and alleviating excessive cross-linking of proteins. Furthermore, ultrasonic promoted protein unfolding even under the protection of trehalose. When added simultaneously, trehalose and TGase weakened their respective effects on protein structure.