Emerging nanomedical strategy is to construct a nanoagent that affords not only diagnostic and therapeutic functions but also imaging-guided treatment. It is crucial to understand the in vivo biological processes of nanoagents for improving theranostic function and biosafety. Herein, we report a multimodal photoacoustic/single-photon emission computed tomography (SPECT) imaging technique to dynamically monitor the in vivo behaviors of nanoagents. Near-infrared cypate-induced silk fibroin nanoassembly was chosen as the nanoagent object due to their promise in biocompatibility and aggregation-enhanced photothermal effect. This unique effect makes the nanoagents useful for the integration of photoacoustic imaging and photothermal therapy. Moreover, the nanoagents are also labeled with the radionuclides (99mTc) to render SPECT imaging. Multimodal photoacoustic/SPECT imaging provides real time, noninvasive, sensitive, and whole-body 3D information about nanoagents' distribution in vivo. These results highlight the significance of visualizing the in vivo behaviors of nanoagents and locating the tumor in vivo, substantially benefiting the better treatment planning.