Estradiol is a critical hormone for reproductive health in females and males both. Monitoring estradiol levels can aid in diagnosing various conditions such as menopause, infertility and even some cancers. Estradiol is also a type of endocrine-disrupting chemical (EDC) that has diverse impacts on ecosystems as well as human health. In can enter the environment through agricultural runoff, sewage and industrial effluents. Adapting both perspectives, establishing biosensors for estradiol detection becomes crucial. In this study, 17-α-ethinylestradiol imprinted polymeric nanoparticles (17EE-MIPs) were synthesized using mini-emulsion polymerization and characterized. Prior to consecutive Quartz Crystal Microbalance (QCM) based analysis, 17EE-MIPs were tested with Surface Plasmon Resonance (SPR), from which the equilibrium and binding kinetic analysis as well as equilibrium isotherm models were derived. Freundlich model was found to best represent the 17EE-MIP based SPR platform. Next, 17EE-MIPs were covalently attached on the QCM crystal and different 17EE concentrations were tested consecutively without regeneration steps. Both systems yielded very high linearity with R2 values of 0.9798 and 0.9895 for SPR and QCM, respectively. The limit of detection (LOD) of the SPR and QCM sensor were calculated as 11.57 and 1.335 µM, respectively. Here, two sensing platforms were employed to crosscheck the performance of the 17EE-MIPs, both verified to respond to low concentrations of 17EE with high consistency.