Highly soluble fluorescent pyrene derivative with substantially improved fluorescence intensity in aqueous buffer was obtained via PEGylation strategy. The highly soluble PEGylated pyrene (PEO-Py) non-covalently adsorbed onto the surface of gold nanoparticles (Au NPs) to form dyads with quenched fluorescence due to highly efficient energy transfer between PEO-Py and Au NPs. The PEO-Py/Au NPs dyads were used for the sensitive turn-on fluorescent detection of biothiols. The fluorescence of PEO-Py was restored by the addition of cysteine (Cys), indicating that Cys can modulate the energy transfer between PEO-Py and Au NPs. This phenomenon then allowed for the sensitive detection of Cys with a limit of detection (LOD) of 11.4 nM. The linear range of determination of Cys was from 1.25 x 10(-8) to 2.25 x 10(-7) M. None of the other amino acids found in proteins showed obvious interference with the determination. It was important to note that the detection sensitivity of the PEO-Py/Au NPs system was more than 5-fold improved compared with the Py/Au NPs system. In addition, other biothiol molecules, such as glutathione, could also be detected by this sensor system. The method was also successfully applied to the determination of the total content of aminothiols in human plasma. Therefore an easily prepared, inexpensive, high solubility fluorescent probe has been realized and is also expected to detect other biological analytes of interest.