A multifunctional nanoparticle for biological applications has been developed by combining the useful functions of quantum dots (QDs), fluorescent dyes, and organosilica particles. Dual fluorescent thiol-organosilica nanoparticles composed of QDs and a fluorescent dye in a thiol-organosilica layer were synthesized in a one-pot process. The thiol-organosilica layer encapsulating the QDs could be tuned in thickness and functionalized. The QDs encapsulated with thiol-organosilica layer (thiol-OS-QDs) were well dispersed, and exhibited unique photophysical properties upon encapsulation within the thiol-organosilica layer. The thiol-OS-QDs could preserve their fluorescent properties under severe acid and alkali conditions as compared with those of bare QDs. The dual fluorescent thiol-OS-QDs containing QD605 and rhodamine B showed strong fluorescence, high photostability, and reduced photoblinking. Cells labeled with dual fluorescent thiol-OS-QDs were detected clearly in vivo, and their functionalization with antibody using the thiol residue on the surface was useful for molecular tracing of tumor antigen. The dual fluorescent thiol-OS-QDs showed high potential as non-photoblinking QDs.