The in situ electrodeposition of cholesterol oxidase (ChOx)-modified polydopamine (PDA) thin film on nanostructured-screen printed electrodes (SPEs) is described here. Prussian Blue (PB) and multi-wall carbon nanotubes (MWCNTs) were used for their outstanding properties in biosensing applications. Cyclic Voltammetry (CV), Constant Potential Amperometry (CPA) and Electrochemical Impedance Analysis (EIS) were used to confirm the correct electrodeposition of the enzyme -modified PDA thin film on the SPE. In addition, the assembly protocol was fully revised to optimize the biosensor response. The proposed cholesterol biosensor showed Michaelis-Menten behavior with K-M(app), V-max and Hill parameter values of 1.97 mM, 7.33 mu A and 1.06, respectively. Biosensor sensitivity, stability, reproducibility, repeatability and anti-interference properties were obtained and confirmed the feasibility of using this biosensor for cholesterol determination. In order to show the applicability of such an approach, the biosensor was incorporated in a Flow Injection Analysis (FIA) device and had a high sensitivity of 32.14 mA M-1 cm(-2) (R-2 = 0.999), a linear range over two orders of magnitude and a detection limit (S/N = 3) of 1.5 mu M. The results confirmed the success of the biosensor for determining cholesterol in future applications in diagnostic laboratories.