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Electrochemical detection is a promising technique for bio-analytes sensing because of rapid analysis, good selectivity, reusability, thermal stability, linearity and relatively low cost. There have been consistent efforts to improve the performance of the electrochemical biosensors which include functionalization of the working electrode surface by polymer or nanomaterials. In this work, a cholesterol sensor is developed based on carbon nanotube electrode with electrochemical polymerized enzyme immobilization First, CNTs are selectively grown on 1 mm2 window of gold coated SiO2/Si substrate by thermal chemical vapor deposition (CVD) with water-assisted etching. CNTs are then simultaneously functionalized and enzyme immobilized by electrochemical polymerization of polyaniline and cholesterol enzymes. Scanning electron microscopic characterization reveals polymer-enzyme nanoparticle coating on CNT surface. Fourier transform infrared spectroscopy is then used to confirm that the coated nanoparticles are polyaniline-cholesterol enzyme composite. The fabricated sensors have been characterized for cholesterol detection in the concentration range between 0 to 300 mg/dl by standard cyclic voltammogram measurement. CV curves show the oxidation and reduction peaks centered around 450 and -220 mV, respectively. In addition, the peaks become clearly pronounced and shifted toward lower voltage as the cholesterol concentration increases. An almost linear relationship between the cholesterol concentration and the response current could be observed in the concentration range of 100-300 mg/dl with good sensitivity. Thus, the fabricated bio probe is effective for cholesterol detection in normal range of cholesterol concentration in human blood.