A new type of fluorescence “off-on” probe was designed for L-Cysteine (L-Cys) based on the fluorescence resonance energy transfer (FRET) between negatively charged amino-capped porous silicon nanoparticles (SiNPs) and positively charged citrate-stabilized Au nanoparticles (AuNPs). In this proposed FRET immunosensor, novel water-soluble amino-conjugated porous SiNPs in ethanol with excellent photoluminescence properties act as the energy donor. Excellent quenching efficiency between SiNPs-ethanol and citrate-stabilized AuNPs by electrostatic interaction via FRET provides an ideal “off-state” (turn-off). The addition of L-Cys leads to releasing the adsorbed AuNPs from the surface of SiNPs and hence the fluorescence emission of SiNPs-ethanol is restored (turn-on), which means the coordination ability of the thiols with AuNPs is stronger than that of the electrostatic interaction. The fluorescence intensity of SiNPs-AuNPs in ethanol is sensitive to L-Cys, and such a restored fluorescence is proportional to the concentration of L-Cys. The method will broadly benefit the development of a new thiol biosensor based on nanostructured porous materials, and the proposed procedure is also expected to develop a variety of functional nanoparticles to form other novel kinds of nanosensors.