In recent years, biosynthesis of silver nanoparticles using plant extracts and bioactive compounds has gained much attention. Green synthesis of nanoparticles is eco‐friendly, cost‐effective, and is a promising substitute for the chemical synthesis of silver nanoparticles. Also, the previous experiments have revealed that plants raise the cytotoxicity potentials of silver nanoparticles against several cell lines especially tumor cell lines. The present study confirms the potential of aqueous extract of Glycyrrhiza glabra L leaf grown under in vitro condition for the biosynthesis of silver nanoparticles (AgNPs). Also, in this study, we revealed the antioxidant and anti‐acute T cell leukemia properties of AgNPs against J45.01, Jurkat, Clone E6–1, J.CaM1.6, and J.RT3‐T3.5 cell lines. Silver nanoparticles were characterized and analyzed using common nanotechnology techniques including UV–Vis. and FT‐IR spectroscopy, TEM, and FE‐SEM. DPPH free radical scavenging test was carried out to assess the antioxidant potentials of AgNO3, G. glabra, and AgNPs. MTT assay was used on normal cell line (HUVEC) and acute T cell leukemia cell lines (J45.01, Jurkat, Clone E6–1, J.CaM1.6, and J.RT3‐T3.5) for analyzing of anti‐acute T cell leukemia properties of AgNO3, G. glabra, and AgNPs. FT‐IR analysis revealed antioxidant compounds in the nanoparticles were the sources of reducing power, reducing silver ions to AgNPs. TEM and FE‐SEM images indicated a uniform spherical morphology in size of 20 nm for silver nanoparticles. DPPH test indicated similar antioxidant activities for G. glabra, AgNPs, and butylated hydroxytoluene. Silver nanoparticles had very low cell viability dose‐dependently against J45.01, Jurkat, Clone E6–1, J.CaM1.6, and J.RT3‐T3.5 cell lines without any cytotoxicity on HUVEC cell line. The best result of cytotoxicity property of AgNPs against above cell lines was seen in the case of J.CaM1.6 cell line. Above findings confirm the significant antioxidant and anti‐acute T cell leukemia properties of AgNPs. After confirming these results in clinical trial studies, AgNPs can be used as a chemotherapeutic drug for the treatment of acute T cell leukemia in human.