Background: Tephrosia villosa (Leguminosae) is an important medicinal plant widely used in traditional Indian medicine. To the best of our knowledge, there is no report on this field. Objective: The aim of this study was to synthesize silver nanoparticles (SNPs) using T. villosa in vivo leaf and in vitro callus extract and characterization of SNPs. Materials and Methods: The synthesized SNPs were characterized by ultraviolet (UV) visible spectrum, Fourier transform infrared (FT-IR), X-ray diffraction (XRD), scanning electron microscopic (SEM), and Energy-dispersive X-ray spectroscopy (EDX). The in vitro antioxidant activities of the synthesized nanoparticles were studied by 1, 1-diphenyl-2-picryl-hydrazyl radical scavenging activity, hydrogen peroxide assay, nitric oxide radical scavenging activity, and hydroxyl radical scavenging activity. Results: The in vivo leaf SNPs have absorbance peak at 740 nm, and callus SNPs have absorbance peak at 305 nm for reaction at room temperature. The FTIR results also showed interaction between the plant extract and Ag-NPs due to the similarity in the peak patterns. The four and nine diffraction peaks were obtained from leaf and callus SNPs, respectively, through XRD studies. The obtained SEM image confirms the presence of polymorphic nanoparticles such as spherical, ellipsoidal, and some irregular shaped. The EDX results showed that Ag-NPs display an absorption peak at 3 keV, indicating the presence of the element silver. Conclusion: It is confirmed that both the in vivo leaf and in vitro callus SNPs revealed high level of antioxidant activity in in vitro system.