Phytomedicine research received tremendous attention for novel therapeutic agent due to their safety and low cost. We assessed a novel nanoformulation of Biophytum sensitivum (BS), natural flavonoids for their improved efficacy and superior bioavailability against crude extract for prostate cancer cells (PC3). We prepared a nanomedicine of BS by nanoprecipitation method and size analysis via DLS and SEM revealed a range of 100-118 nm and surface zeta potential as −9.77 mV. FTIR was performed to evaluate functional for presence of carbonyl and aromatic rings, respectively. Human PC3 cells showed concentration at 0.5, 0.8, and 1 mg/ml dependent cytotoxicity 22, 39, and 56% for 24 h, whereas 43, 41, and 67% for 48 h of BS nanomedicine compared with crude 11, 22, and 53% for 24 h and 38, 31, and 60% for 48 h, respectively. Haemocompatibility of BS nanomedicine at the concentration of 0.5, 0.8, and 1 mg/ml did not show blood aggregation. Cellular uptake was confirmed using rhodamine-conjugated BS nanomedicine for 48 h. Interestingly, BS nanomedicine 1 mg/ml decreases the nitrite productions in PC3 cells. Collectively, BS nanomedicine has the potential anti-cancer agents with biocompatible and enhanced efficacy can be beneficial for the treatment of prostate cancer