We report the first attempt to use a naturally occurring biopolymer, such as humic acid (HA), as modifiers to prepare a new organicinorganic composite film with the 3-n-propylpyridiniumsilsesquioxane chloride Pt nanoparticles (Pt-SiPy+Cl-). By using layer-by-layer (LbL) technique, we have constructed multilayered films, (HA/Pt-SiPy+Cl-)(n) and (Pt-SiPy+Cl-/HA)(n), with adjustable thickness due to negatively charged macromolecule HA, which assists the electrostatic interaction with the positively charged of the Pt-SiPy+Cl- nanohybrid. The combination of these partially charged polyelectrolytes in the LbL film was explored by spectroscopic and electrochemical techniques. The formation of the film (HA/Pt-SiPy+Cl-)(n) is more efficient, since the presence of the macromolecule in the inner layer provides a greater number of anchoring sites for the hybrid, ensuring a more linear and uniform growth of the LbL films. This film also presented a better electroanalytical response for oxidation of 17 alpha-ethynylestradiol, EE2, indicating that the Pt nanoparticles strongly influences the electrochemical oxidation of estrogen. The analytical curve for EE2 was linear in the concentration range of 1.37 to 21.4 mu mol L-1 (R = 0.998), with a detection limit of 1.10 mu mol L-1 and quantification limit of 3.68 mu mol L-1. The obtained results showed that the synergism between HA and Pt-SiPy+Cl- improves the electroactive properties of the sensor. (C) 2016 The Electrochemical Society. All rights reserved.