Methylene blue (MB) doped silica nanoparticles (MB-SiNPs) were synthesized in the water-in-oil microemulsion. By optimizing the concentration of MB doped in the silica matrix, the near-infrared fluorescent MB-SiNPs with strong fluorescence were obtained. Then, the feasibility of MB-SiNPs for cell labeling and tracking in vivo was studied. The cell viability assay of Hela cells in the presence of MB-SiNPs was performed by using MTT assay, and the result indicated that, as the concentration of MB-SiNPs was 1 mg/mL in medium, the cell survival percentage was 80%. The distribution of MB-SiNPs in Hela cells was assessed by laser scanning confocal microscopy, and the result showed that MB-SiNPs entered cells easily and mainly located in lysosomes. In vivo fluorescence imaging of MB-SiNPs after tail vein intravenous injection showed that the whole body of the mice emitted NIR fluorescence and most of the MB-SiNPs were accumulated in some organs, such as liver and spleen. All the results demonstrated that MB-SiNPs could be used for cell labeling and in vivo imaging.