We describe a rapid environmentally friendly wet-chemical approach to synthesize extremely stable non-toxic, biocompatible, water-soluble monodispersed gold nanoparticles (AuNPs) in one step at room temperature. The particles have been successfully achieved in just a few minutes by merely adding sodium hydroxide (NaOH) acting as an initiator for the reduction of HAuCl(4) in aqueous solution in the presence of polyvinylpyrrolidone (PVP) without the use of any reducing agent. It is also proved to be highly efficient for the preparation of AuNPs with controllable sizes. The AuNPs show remarkable stability in water media with high concentrations of salt, various buffer solutions and physiological conditions in biotechnology and biomedicine. Moreover, the AuNPs are also non-toxic at high concentration (100 microM). Therefore, it provides great opportunities to use these AuNPs for biotechnology and biomedicine. This new approach also involved several green chemistry concepts, such as the selection of environmentally benign reagents and solvents, without energy consumption, and less reaction time.