By binding molecular probes that target tumor cells, gold nanoparticles (AuNPs) with superior characteristics have shown great potential in tumor molecular imaging studies. The non-invasive, high-resolution, and three-dimensional imaging of the targeted AuNPs within the tumor is desirable for both diagnosis and therapy. In this study, gold nanoflowers (AuNFs) are presented as a novel contrast agent for photoacoustic tomography (PAT). By binding to folic acid, the molecular probe, the tail-vein injected AuNFs concentrated within the tumor site in mice; this was clearly visualized by three-dimensional (3D) PAT imaging. In addition, toxicity assay proved that AuNFs were harmless to living cells and animals. Our results demonstrate that AuNFs have great potential in tumor molecular imaging.