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Eco-friendly, nontoxic, inexpensive, abundantly available hitherto unreported Eclipta prostrata leaf extract is used for the biosynthesis of titanium dioxide nanoparticles (TiO2 NPs). The TiO2 NPs were characterized by FTIR, XRD, AFM and FESEM analysis. FTIR peak implicated the role of carboxyl group O-H stretching amine N-H stretch in the formation of TiO2 NPs. XRD characterized in crystallographic plane of rutile phase. AFM showed uneven surface morphology which indicates the presence of both individual and agglomerated nanoparticles. FESEM analysis showed shape in spherical clusters, quite polydisperse and it ranges in size from 36-68 nm with calculated average size of 49.5 nm. In this paper, we have demonstrated a novel biological route for the synthesis of TiO2 NPs and first report to assess the larvicidal activity against malaria vector.