Herein, a novel nanocomposite of G/PVP/PANI was prepared and used to modify the working electrode of a paper-based biosensor via electrospraying. The droplet-like structures of the G/PVP/PANI-nanocomposite-modified electrode was used for the sensitive determination of H2O2 and cholesterol using amperometry. The performance of this sensing system was optimized and then applied to the determination of cholesterol concentration in a complex biological fluid (e.g., human serum).