The integration of ultrasound (US) contrast enhancement with oxygen‐loading nanoagents provide the synergistic strategy for simultaneously US imaging and hypoxic microenvironment modulation. Herein, we synthesize pentafluorobutane (PFB)‐loading methoxy poly(ethylene glycol)‐b‐poly(l‐lactide) (PLLA) nanoparticle as the novel US‐contrast‐enhanced agent and demonstrate that PFB@PLLA effectively loads oxygen. We characterize the nanosize, phase‐transformation property and oxygen‐loading amount of PFB@PLLA and investigate the effectiveness of these nanoagents in US‐contrast‐enhanced imaging. The PFB@PLLA displays a perfect temperature‐responsive phase‐transition property and its liquid‐to‐gas phase transition temperature is 45°C, which produces microbubbles in the targeted regions. Moreover, PFB@PLLA loads high amount of oxygen and US‐triggering PFB@PLLA reoxygenation effectively inhibits the expression of hypoxia‐related proteins (HIF‐1α and CAIX), reduces lactate secretion and glycolysis, which modulates hypoxic microenvironment and inhibits cancer cell migration and invasion in vitro. This study demonstrates that the US contrast‐enhanced activity of PFB@PLLAs and the promising utility of oxygen‐loading nanoagents to improve hypoxic microenvironment.