The surface modified and aligned mesoporous anatase titania nanofiber mats (TiO2-NF) have been fabricated by electrospinning for esterified cholesterol detection by electrochemical technique. The electrospinning and porosity of mesoporous TiO2-NF were controlled by use of polyvinylpyrrolidone (PVP) as a sacrificial carrier polymer in the titanium isopropoxide precursor. The mesoporous TiO2-NF of diameters ranging from 30 to 60 nm were obtained by calcination at 470 °C and partially aligned on a rotating drum collector. The functional groups such as -COOH, -CHO etc. were introduced on TiO2-NF surface via oxygen plasma treatment making the surface hydrophilic. Cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) were covalently immobilized on the plasma treated surface of NF (cTiO2-NF) via N-ethyl-N0-(3-dimethylaminopropyl carbodiimide) and N-hydroxysuccinimide (EDC-NHS) chemistry. The high mesoporosity (∼61%) of the fibrous film allowed enhanced loading of the enzyme molecules in the TiO2-NF mat. The ChEt-ChOx/cTiO2-NF-based bioelectrode was used to detect esterified cholesterol using electrochemical technique. The high aspect ratio, surface area of aligned TiO2-NF showed excellent voltammetric and catalytic response resulting in improved detection limit (0.49 mM). The results of response studies of this biosensor show excellent sensitivity (181.6 μA/mg dL(-1)/cm(2)) and rapid detection (20 s). This proposed strategy of biomolecule detection is thus a promising platform for the development of miniaturized device for biosensing applications.