This paper reports that the high electrocatalytic activity of RuO2 nanowires grown on electrospun TiO2 nanofibers for the oxidation of L-ascorbic acid (AA); and the application of these materials for direct selective sensing of AA in complex samples. Compared to bare glassy carbon (GC) electrode, RuO2 nanowires on TiO2 nanofibers-loaded GC electrode facilitates the oxidation of AA most drastically among the tested species: AA, 4-acetamidophenol (AP), dopamine (DA), uric acid (UA), and glucose. The amperometric response of RuO2 nanowires on TiO2 nanofibers at the applied potential of 0.018 V (vs. SCE) exhibits high sensitivity (268.2 +/- 3.7 mu A mM(-1) cm(-2), n=5), low detection limit (< 1.8 mu M), great linearity, reasonable stability, and exclusive selectivity over AP, DA, glucose and UA at their physiological levels. In differential pulse voltammetry, it is verified that the potential resolution of RuO2 nanowires on TiO2 nanofibers is able to differentiate AA, DA, UA, and AP one from the others. In addition, as prepared RuO2 nanowires on TiO2 nanofibers are successfully applied for direct and selective AA measurements in commercial vitamin samples and for the real-time direct analysis of AA generated from living rat liver tissue in vitro. (C) 2015 Elsevier B.V. All rights reserved.