17 alpha-ethinylestradiol (EE2) is a female synthetic hormone with a high estrogenic action that can potentially disrupt the organisms' endocrine system, leading to severe health problems. Such fact increases the need for technologies capable of detecting this compound even at very low levels. In this context, here we report on the development of a highly sensitive electrochemical sensor for EE2 detection based on a novel nanoarchitecture composed of electrospun nanofibers of polyvinylpyrrolidone (PVP)/chitosan (Chi)/ reduced graphene oxide (rGO) functionalized with Laccase enzyme. The hybrid nanofibers deposited onto fluorine doped tin oxide (FTO) electrode showed good electrochemical properties with synergistic effect between PVP, Chi and rGO and suitability to assemble the enzyme Laccase. The PVP/Chi/rGO_Laccase modified electrode was used for EE2 amperometric detection showing a very low limit of detection of 0.15 pmol L-1 (3.3 o/S), good reproducibility with relative standard deviation (RSD) equal to 4.29% and 8.44% to intra- and inter-electrode, respectively. In addition, the biosensor was shown to be selective for EE2 detection in the presence of several common interfering compounds. The developed platform was employed for real sample analysis, showing good accuracy towards EE2 determination in synthetic and human urine samples, suggesting the biosensor as a potential tool for the determination of EE2 in varied environments.