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The development of eco-friendly approach for the preparation of monodispersed gold nanoparticles (GNPs) has received much attention for their easy application. Most of the current methods involve known protocols which employ toxic chemicals and hazardous byproducts. This greatly limits their use in biomedical fields, particularly in clinical applications. Recent research has been focused on green synthesis methods to produce different nanoparticles with suitable commercial viability. The biosynthesis of monodispersed GNPs using the spent cultures of Klebsiella pneumoniae as reducing and stabilizing agent has been reported. The gold salt concentration to improve monodispersity and stability of GNPs has been optimized. Synthesized GNPs were characterized by UV–Visible spectroscopy showed absorption spectra in the range of 530–560 nm at different concentrations of HAuCl4. At the optimum reaction concentration of 1.5 mM HAuCl4, absorption peak was obtained at 535 nm. The GNPs have been further characterized by X-ray diffraction, FTIR, DLS and TEM analysis. The DLS graph showed that the particles were more monodispersed. The TEM image showed the formation of spherical shaped GNPs in the range of 4–10 nm. The effect of gold salt concentration on dispersity, size and stability of the biosynthesized GNPs has been reported.