The development of free and total cholesterol nanobiosensors based on a single step electrochemical integration of gold nanoparticles (AuNPs), cholesterol oxidase (COx), cholesterol esterase (CE) and a mediator with polypyrrole (PPy) films is described. The incorporation of the various components in the PPy films was confirmed by chronopotentiometry, cyclic voltammetry (CV), scanning electron microscopy, energy dispersive X-ray analysis (SEM-EDX), and Fourier transformed infrared (FTIR) spectroscopy. The free cholesterol, PPy-NO3 --Fe(CN)6 4--AuNPs-COx, nanobiosensor achieved a minimum detectable concentration of 5 μM, a linear concentration range of 5–25 μM and a sensitivity of 1.6 µA cm−2 µM−1 in 0.05 M phosphate buffer (pH 7.00). For the total cholesterol, PPy-NO3 --Fe(CN)6 4--AuNPs-COx-CE, nanobiosensor which also involved the co-incorporation of cholesterol esterase (CE) with the other components, the achieved performances include a minimum detectable total cholesterol concentration of 25 μM, a broader linear concentration range of 25–170 μM and a lower sensitivity of 0.1 µA µM−1 cm−2. Owing to its high selectivity, the presence of common interferants did not affect the total cholesterol measurement with the PPy-NO3 --Fe(CN)6 4--AuNPs-COx-CE nanobiosensor. Both nanobiosensors were successfully used for direct and indirect determination of total cholesterol in human blood serum samples.