Tissue imaging via fluorescence has been widely used for many years. It stands out due to high sensitivity, high specificity, low cost, use of non-ionizing radiation, capability of multiplex imaging and providing tissue structural, functional and molecular information. However, it suffers from limitations: poor spatial resolution (i.e., fluorescence diffuse optical tomography) or small penetration depth (i.e., fluorescence microscopy) due to the strong tissue light scattering. Thus, high-resolution fluorescence imaging in deep tissue has been sought after for many years. Recently, we developed a new imaging technique—ultrasound-switchable fluorescence (USF) which has been demonstrated to overcome the limitations. We successfully imaged centimeter-large mammary tumors in live mice with much higher spatial resolution than conventional 2D planar fluorescence imaging or 3D fluorescence diffuse optical tomography and demonstrated the capability of USF for high-resolution in vivo imaging. We also compared USF images with micro-CT to further validate the technology. In this talk, I will introduce USF and demonstrate recently acquired images from in vitro and in vivo deep tissues. Potential biomedical applications of USF in different fields will be discussed.