Highly monodisperse, dendritic, and functionalized mesoporous silica nanospheres (MSNs) with sub-200 nm size were synthesized in a one-pot sol-gel reaction, by a dual-templating micelle system consisting of a partially fluorinated short-chain anionic fluorocarbon surfactant and cetyltrimethylammonium bromide. This kind of anionic fluorocarbon surfactant works simultaneously as a swelling agent to enlarge the pore of the MSNs, an ion-pair agent to the structure-directing silane in the preparation of amine-functionalized MSNs, and a surface tension reducing agent to make the system thermodynamically more stable for producing more uniform MSNs. The particle size and the morphology of the resultant MSNs can be fine-tuned by changing the amount of the fluorocarbon surfactant added and the ratio of the functional group containing organosilane to tetraethoxysilane. Subsequently, the as-prepared MSNs were used as base materials for the preparation of drug delivery nanomaterials through the surface grafting of a pH-sensitive drug-conjugated polymer and fluorescent nanomaterials through the embedding of europium(III) complex or the immobilization of large molecule fluorescein isothiocyanate-bovine serum albumin.