Blood coagulation (Blood clotting) has emerged as a dangerous consequence associated with coronavirus disease, hypercholesteremia, heart failure, obesity, and smoking. Therefore, the utilization of medicinal plants containing anti-coagulant compounds and biologically safe has become imperative. Lagerstroemia tomemtosa characterized by having polyphenols which have highly antioxidant activity and may have a thrombolytic activity. Biotechnological techniques play a crucial role in the enhancement of the selective compounds. The main object is the production and scaling up of target compounds with anticoagulant effects using biotechnological techniques. Callus production and hairy root culture and evaluating their activity through in-vivo study. Seeds were used for the production of calli and cell suspension cultures. Hairy root culture was established by Agrobacterium rhizogenes bacteria. Ethanol extracts for cell suspension culture and hairy root culture were prepared. Moreover, crude ethanol (70%) and polar and non-polar extracts were prepared from leaves for the comparative study. Chemical analysis was performed on all prepared extracts using HPLC, along with in-vitro antioxidant evaluation by three assays and in-vivo anticoagulant activity. MS medium fortified with 1 mg/L BA+ 0.1 mg/L NAA is the best medium for calli production and cell suspension cultures. The system of hairy root biotransformation was established. The major compounds were separated and identified from crude extract. Hairy root extract showed the highest value of total phenolic and total flavonoid contents either quantitatively or qualitatively. All extracts had antioxidant activity, but the hairy root extract showed the highest activity (IC50; 11.70 ug/ mL). Also, it exposed potent anti-inflammatory and anticoagulant activity more than the crude, non-polar, and polar extracts via intrinsic and/or extrinsic pathways and was safer than warfarin medication. Five major compounds were isolated and identified as ellagic acid, gallic acid, catechin, vitexin, and isovitexin. In conclusion, biotechnological studies achieved the goal of the enhancement of the phenolic constituents and, thus, their activity. Lagestromia tomentosa is suggested to be used as a potent anticoagulant drug.