A FRET-based nanoprobe consisting of CdTe quantum dot as donor and rhodamine B as acceptor embedded in gelatin nanoparticles was constructed and the photophysics of the system was characterized. The FRET-based sensors were led to at fixed donor/acceptor distance by synthesizing via a two-step desolvation way. This approach allowed for short acceptor-donor separation and thus for high energy transfer efficiencies. The morphology of the nanoprobes was presented spherically and relatively uniform with a diameter of about 100 nm, which was characterized by transmission electron microscope. The luminescent property was proved to be stable in vitro and in living cells, indicating that the nanoprobes can be expected to be promising candidates for biological imaging studies.