In this work, we report the development of a simple and sensitive sensor based on graphite screen-printed electrodes (GSPEs) modified by a nanocomposite film for dopamine (DA) detection. The sensor was realized by electrodepositing polyaniline (PANI) and gold nanoparticles (AuNPs) onto the graphite working electrode. The sensor surface was fully characterized by means of the cyclic voltammetry (CV) technique using [Fe(CN)6]4−/3− and [Ru(NH3)6]2+/3+ as redox probes. The electrochemical behavior of the nanocomposite sensor towards DA oxidation was assessed by differential pulse voltammetry (DPV) in phosphate buffer saline at physiological pH. The sensor response was found to be linearly related to DA concentration in the range 1–100 μM DA, with a limit of detection of 0.86 μM. The performance of the sensor in terms of reproducibility and selectivity was also studied. Finally, the sensor was successfully applied for a preliminary DA determination in human serum samples.