A sensitive impedimetric immunosensor was constructed by using an electropolymerized nanocomposite film containing polypyrrole (PPy), polypyrrolepropylic acid (PPa), and Au nanoparticles. The nanocomposite exhibits good stability, high porosity, high hydrophilicity, and efficient probe immobilization capability. In the film, PPa enhances the hydrophilicity while providing covalent probe attachment linkers, PPy promotes the conductivity and electroactivity, and Au nanoparticles result in good conductivity, high stability, and covalent binding linkers. These combined advantages significantly improve the detection sensitivity in comparison to the conventional methods. As a model, a human interleukin 5 (IL-5) immunosensor, an important sensor for disease pathology study, clinic diagnosis, and pharmaceutical research, was fabricated with the new nanocomposite film. Various optimization works were conducted to improve the detection sensitivity. With the optimal fabrication parameters, the detection limit for IL-5 was 10 fg/mL in phosphate buffered saline (PBS) and 1 pg/mL in 1% human serum with good specificity and a dynamic range of 3 orders of magnitude. This work demonstrates a new approach to develop a sensitive and labeless impedimetric immunosensor for potential broad applications in clinical diagnosis and drug discovery.