This investigation aimed to study the antioxidant and anti-inflammatory effects of the aqueous extract of Medicgo sativa and copper nanoparticles CuNPs. Biosynthesized CuNPs were characterized by analytical methods. Qualitative phytochemical analysis was carried out by standard protocols. Quantitative Phytochemical analyses were analyzed by using HPLC methods. Moreover, in vivo intraperitoneally acute toxicity testing of nanoparticles was carried up. Results of qualitative phytochemical and quantitative HPLC analysis results revealed that M. sativa contains most of bioactive compounds, especially Naringin, Rutin, and Vanillic Acid compounds with high antioxidant and anti-inflammatory activity. Characterization of CuNPs confirmed the involvement of biological molecules in CuNPs synthesis with size ranged from 19.8 to 92.8 nm. In this study, the intraperitoneal toxicity test showed no mortality and minor behavioral variations up to 20 mg/kg of CuNPs in Wistar rats. We concluded that M. sativa L has potential properties as biocatalyst stabilizers for CuNPs synthesis and MsE-CuNPs revealed good activity as a potent antioxidant, and anti-inflammatory agent. Further in vivo studies are needed to explore them as good therapeutic agents.