The biodistribution and urinary excretion of different surface-modified silica nanoparticles (SiNPs) in mice were investigated in situ using an in vivo optical imaging system. Three types of surface-modified SiNPs, including OH-SiNPs, COOH-SiNPs, and PEG-SiNPs with a size of approximately 45 nm, have been prepared with RuBPY doped for imaging purposes. Intravenous (i.v.) injection of these SiNPs followed by fluorescence tracing in vivo using the Maestro in vivo imaging system indicated that OH-SiNPs, COOH-SiNPs, and PEG-SiNPs were all cleared from the systemic blood circulation, but that both the clearance time and subsequent biological organ deposition were dependent on the surface chemical modification of the SiNPs. Thus, for instance, the PEG-SiNPs exhibited relatively longer blood circulation times and lower uptake by the reticuloendothelial system organs than OH-SiNPs and COOH-SiNPs. More interestingly, in vivo real-time imaged dominant signal in bladder and urine excretion studies revealed that all three types of i.v.-injected SiNPs with a size of approximately 45 nm were partly excreted through the renal excretion route. These conclusions were further confirmed through ex vivo organ optical imaging and TEM imaging and energy-dispersed X-ray spectrum analysis of urine samples. These findings would have direct implications for the use of SiNPs as delivery systems and imaging tools in live animals. Furthermore, our results demonstrate that the in vivo optical imaging method is helpful for in vivo sensing the biological effects of SiNPs by using luminescent dye doped in the silica matrix as a synchronous signal.