A photoacoustic nanoprobe is self-assembled from human serum albumin and two types of dye molecules, one is inert to pH and the other is pH sensitive. This probe and the quantitative ratiometric photoacoustic pH imaging method are shown to have high safety, be easy-to-operate, and have depth-independent accuracy for real-time in vivo pH imaging of entire tumors. These features make them promising for future cancer prognosis and therapeutic planning.