The synthesis of metallic nanoparticles has become an important issue in recent decades due to their various beneficial and unique properties and biomedical and industrial applications 1. Nanotechnology is seeking to synthesize nanoparticles have substantial biomedical applications because of their unique characteristics and their green nature due to using some medicinal plants as ABSTRACT The object of this work is biosynthesizing AgNPs from extracts of Cyperus sp. galingale rhizomes, studying their characteristics using UV-visible spectroscopy, AFM, SEM, FTIR, and EDX analyses and testing their anticancer activity (in vitro) against L20B cell line. Biosynthesizing AgNPs using various plants is considered eco-friendly, cheap, energy saving and reproducible compared with non-green methods. UV-Visible spectrum checked the surface plasmon resonance of AgNPs at 410-420 nm. FT-IR exhibited that the presence of carbonyl and hydroxyl groups in the extract of Cyperus sp. can reduce and stabilize AgNPs. EDX, SEM, and AFM analyses were applied to confirm the nature, morphology and topography of the biosynthesized AgNPs. AgNPs are spherical or irregular in shape with the average diameter of hot extract-AgNPs is 56.31 nm in comparison with cold extract-AgNPs is 92.53 nm. The hot extract-AgNPs paly a suitable role against mouse cell line (L20B) which have receptors for polioviruses better than the cold extract-AgNPs.