Despite sufficient studies performed in non-primate animal models, there exists scanty information obtained from pilot trials in non-human primate animal models, severely hindering nanomaterials moving from basic research into clinical practice. We herein present a pioneering demonstration of nanomaterials based optical imaging-guided surgical operation by using macaques as a typical kind of non-human primate-animal models. Typically, taking advantages of strong and stable fluorescence of the small-sized (diameter: ~ 5 nm) silicon-based nanoparticles (SiNPs), lymphatic drainage patterns can be vividly visualized in a real-time manner, and lymph nodes (LN) are able to be sensitively detected and precisely excised from small animal models (e.g., rats and rabbits) to non-human primate animal models (e.g., cynomolgus macaque (Macaca fascicularis) and rhesus macaque (Macaca mulatta)). Compared to clinically used invisible near-infrared (NIR) lymphatic tracers (i.e., indocyanine green (ICG); etc.), we fully indicate that the SiNPs feature unique advantages for naked-eye visible fluorescence-guided surgical operation in long-term manners. Thorough toxicological analysis in macaque models further provides confirming evidence of favorable biocompatibility of the SiNPs probes. We expect that our findings would facilitate the translation of nanomaterials from the laboratory to the clinic, especially in the field of cancer treatment.