In recent years, silica coating has been extensively investigated to fabricate the biocompatible interface of quantum dots (QDs) for biomedical applications. We here describe a facile and efficient method of synthesizing high-quality silica-coated CdSeS QDs (CdSeS QD/SiO2), where an immediate photoluminescence-favorable microenvironment is first created by assembling amphiphilic molecules around the CdSeS core, and a thin silica shell is further introduced to protect this hydrophobic interlayer, The prepared CdSeS QD/SiO2 exhibits excellent properties such as good water solubility, low cytotoxicity, and high quantum yield (QY, up to 0.49) as well as the resistance of photobleaching in aqueous solution. Also, the CdSeS QD/SiO2 nanoparticles homogeneously comprise single CdSeS cores and hold a comparatively small size up to about 11 nm in diameter. Particularly, this method leads to a significant increase in QY as compared to the uncoated CdSeS QDs (similar to 109%, of the initial QY), though only thin silica shells formed in the CdSeS QD/SiO2 structure. By coupling with folic acids, the CdSeS QD/SiO2 conjugates were successfully used for tumor cell labeling. Our results demonstrated a robust hydrophobic QDs-based approach for preparing highly photoluminescent, biocompatible QD/SiO2 through creation of a stable hydrophobic interlayer surrounding the QD cores, which could be also suitable for silica coating of other kinds of hydrophobic nanoparticles.