Background: The production of nanoparticles using a biosystem is considered green chemistry. Application of plant extracts as a biological process has been proven to be suitable for synthesis of nanoparticles.Objectives: This study designed in order to evaluate the production of silver nanoparticles using Juglans Regia leaf extract and to compare the outcome of different preparation methods of plant extracts (ethanolic extract, boiling water extract and plant powder) for the generation of nanoparticles.Materials and Methods: The reaction mixture contained the following ingredients: AgNO3 (10 mM) as the biotransformation substrate, plant extract or powder as the biocatalyst, glucose (560 mM) as the electron donor, phosphate buffer (pH=7, 100 mM) and ethanol 70% as the solvent in the reaction mixture. The samples were taken from the reaction mixtures at different times, and the absorbance (450 nm) of the colloidal suspensions of silver nanoparticle hydrosols was recorded immediately following dilution (1:80) so as to preserve its freshness.Results: UV-visible spectrophotometer analysis revealed that the direct application of powder of the walnut leaf was the most efficient technique. TEM (Transmission electron microscopy) micrograph obtained by using this method revealed the generation of aggregated polydisperse, quasi-spherical nanoparticles in sizes of 10-50 nm. Ethonolic extract resulted in single silver nanoparticles which were nearly monodisperse, spherical, and individual nanoparticles ranged in size from 1-5 nm. Therefore, using direct powder of Walnut created more particles but applying ethanolic extract synthesized particles with smaller dimensions and no aggregation.Conclusions: Different preparation methods of Juglans Regia influence silver nanoparticles formation.