We prepared a series of well-defined polyfluorene-based copolymers by using polyfluorene (PF) macroinitiators to initiate atom transfer radical polymerization (ATRP) of γ-methacryloxypropyltrimethoxysilane (MPS). The chemical structures of hydroxy-functionalized PF (PFOH), PF macroinitiators (PFBr), and polyfluorene-b-poly(γ-methacryloxypropyltrimethoxysilane) (PF-b-PMPS) block copolymers were confirmed by 1H NMR. In THF/aqueous solution, amphiphilic PF-b-PMPS block copolymers can self-assemble into core-shell micelles with PMPS as the core and PF as the shell. Base-catalyzed sol-gel process inside the PMPS core results in PF-encapsulated silica core-shell nanospheres. Transmission electron microscopy (TEM) studies reveal the dense grafting of PF on the surface of silica core. The effect of PF-b-PMPS core-shell structures on the optical and electrochemical properties was also investigated. This novel kind of luminescent nanosphere has potential applications in bioanalysis, biomedical, and light-emitting devices.