Ultrasound (US) has been applied in clinical practice for its non-invasive and high selectivity. However, it is difficult to achieve a satisfactory anti-tumor effect with US alone. Meanwhile, the use of US therapy alone can exacerbate tumor hypoxia. In this study, we prepared hypoxia-activated 6-diazo-5-oxo-L-norleucine (DON) prodrug nanoparticles (HDON-NPs) to improve US therapeutic effects. In an H22 murine liver cancer model, US therapy selectively disrupted tumor blood vessels, leading to increased tumor hypoxia and a 1.67-fold increase in the expression of nitroreductase (NTR). The combination therapy of US and HDON-NPs demonstrated a synergistic effect, resulting in a tumor suppression rate (TSR) of 90.2% ± 6.4%, which was 5.93-fold higher than that of US therapy alone. The combined treatment selectively blocked the glutamine metabolism of the tumor cells while simultaneously activating the T cells in the tumor microenvironment, thereby exerting a robust anti-tumor effect.