A bifunctional nanoparticles-based carrier for simultaneous in vivo imaging and photodynamic therapy by encapsulating methylene blue (MB) alone in the phosphonate-terminated silica matrix has been developed. The phosphonate-terminated silica nanoparticles, entrapping water-soluble photosensitizer MB (MB-encapsulated PSiNPs), are synthesized by the controlled synchronous hydrolysis of tetraethoxysilane and trihydroxyl silyl propyl methyl phosphonate in the water-in-oil microemulsion. The resulting MB-encapsulated PSiNPs effectively prevent the leakage of entrapped MB from the particles and provide protection for against reduction by diaphorase. Enough dose of irradiation to the MB-encapsulated PSiNPs under the light of 635 nm results in efficient generation of singlet oxygen and induces photodynamic damage to Hela cells. Furthermore, the non-invasive visualization of MB-encapsulated PSiNPs in mice under the in vivo imaging system confirmed the MB-encapsulated PSiNPs also presents near-infrared luminescence for in vivo imaging. And the effect of the PDT toward the xenograft tumor in vivo is exciting after imaging the MB-encapsulated PSiNPs injected tumor using in vivo optical imaging system. Thus, the single particle platform is effective for simultaneous in vivo imaging and photodynamic therapy without using extra agent, which can provide image-guidance for site-specific photodynamic therapy.