A biosensor chip is developed for the detection of a protein biomarker of endocrine disrupting compounds, vitellogenin (Vg) in aquatic environment. The sensor chip is fabricated by immobilizing anti-Vg antibody on 4-Aminothiophenol (4-ATP) coated nanosculptured thin films (nSTFs) of silver on Si substrates. The biosensor is based on the SERS of 4-ATP, enhanced by the Ag nSTFs. Before the fabrication of the sensor, the performance of the enhancement is optimized with respect to the porosity of nSTFs. Further, the biosensor is developed on the nSTF with optimized enhancement. The SERS signals are recorded from the sensor chip for varying concentrations of Vg. A control experiment is performed on another similar protein Fetuin to confirm the specificity of the sensor. The repeatability and reusability of the sensor, along with its shelf life are also checked. The limit of detection of the sensor is found to be 5 pg mL −1 of Vg in PBS within our experimental window. Apart from high sensitivity, specificity and reusability, the present sensor provides additional advantages of miniaturization, requirement of very small volumes of the analyte solution (15 μL) and fast response as compared to conventional techniques e.g., ELISA, as its response time is less than 3 minutes.