Biological effects of rutin bioactive are limited due to its poor oral bioavailability and its degradation in aqueous environments. For the purpose of bioenhancement, different nanoemulsion systems of rutin were developed by aqueous titration method using water as dispersion media. The nanoemulsion systems were characterized for surface morphology, droplet size, polydispersity index, zeta potential, in vitro release profile and the formulations were optimized. The anticancer potential of optimized nanoemulsion was evaluated by cells viability (MTT) assay, nuclear condensation, and ROS activity using human prostate cancer (PC3) cell line. On the basis of cell viability data the inhibitory concentration (IC 50) value for optimized nanoemulsion formulation on PC3 cancer cells was found to be 11.8 μM. Fluorescent microscopic analysis and intracellular ROS generation demonstrated significant ROS induction that might lead to triggering the apoptosis pathway. In conclusion, developed nanoemulsion displayed significant efficacy against prostate carcinoma cells.