Silver nanoparticles (Ag NPs) are appealing due to their excellent antibacterial/antivirus properties. At the meantime, the wide applications of Ag NPs as antibacterial/antivirus agents arise the concern of Ag NPs’ toxicity. However, quantitative understanding of the cytotoxicity of Ag NPs is minimum since that the Ag NPs in current studies have wide size distributions, in which the size effect of Ag NPs on cytotoxicity was unable to be accurately evaluated. In this work, unprecedentedly monodispersed Ag NPs with sizes of 25, 35, 45, 60 and 70 nm were obtained, respectively, by using an optimized polyol method with poly(vinyl pyrrolidone) (PVP) as surfactant. It was found that the reaction temperature, reaction time, concentration of the surfactant and reactants are playing important roles in determining the size and size distribution of Ag NPs. With the monodispersed Ag NPs as standard samples, the size- and dose- dependent cytotoxicity of Ag NPs against Human lung fibroblast (HLF) cells was accurately accomplished in terms of cell viability, apoptosis and necrosis, reactive oxygen species, etc. We expect that the monodispersed Ag NPs will act as the standard samples for quantitatively characterizing the toxicity of Ag NPs in vitro and in vivo.