We present a novel and efficient nanoporous microfluidic biochip consisting of a functionalized chitosan/anatase titanium dioxide nanoparticles (antTiO2-CH) electrode integrated in a polydimethylsiloxane (PDMS) microchannel assembly. The electrode surface can be enzyme functionalized depending on the application. We studied in detail cholesterol sensing using the cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) functionalized chitosan supported mesoporous antTiO2-CH microfluidic electrode. The available functional groups present in the nanoporous antTiO2-CH surface in this microfluidic biochip can play an important role for enzyme functionalization, which has been quantified by the X-ray photoelectron spectroscopic technique. The Brunauer-Emmett-Teller (BET) studies are used to quantify the specific surface area and nanopore size distribution of titania nanoparticles with and without chitosan. Point defects in antTiO2 can increase the heterogeneous electron transfer constant between the electrode and enzyme active sites, resulting in improved electrochemical behaviour of the microfluidic biochip. The impedimetric response of the nanoporous microfluidic biochip (ChEt-ChOx/antTiO2-CH) shows a high sensitivity of 6.77 kΩ (mg dl-1)-1 in the range of 2-500 mg dl-1, a low detection limit of 0.2 mg dl-1, a low Michaelis-Menten constant of 1.3 mg dl-1 and a high selectivity. This impedimetric microsystem has enormous potential for clinical diagnostics applications. This journal is © The Royal Society of Chemistry.