Changes in isoforms of Tau protein, which are critical for microtubule functioning, are accepted as being responsible for diseases characterized by dementia, in particular Alzheimer's disease (AD). In this comprehensive study, a single-use neuro-biosensing probe for the determination of Tau-441 protein was developed by utilizing the power of nanocomposites consisting of reduced graphene oxide (rGO) and gold nanoparticles (AuNP) using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The nanocomposite surface (rGO-AuNP) was modified with 11-mercaptoundecanoic acid (11-MUA) act as covalent anchorer to increase the sensitivity of the assay. Surface coverage value and pinhole ratio were calculated using EIS data. Kramers-kronig data, which helps to interpret instrumental errors, are also calculated. The immunoreaction of Tau-441 with anti-Tau was monitored simultaneously with Single Frequency Impedance (SFI). The changes in surface morphology were evaluated with scanning electron microscopy (SEM), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR). The designed immunosensor showed a linear response within the concentration range of 1-500 pg/mL for the target analyte Tau-441 and the limit of detection was found to be 0.091 pg/mL. The promising point of the study is that this neuro-biosensor system can capture the Tau-441 target protein in both serum fluid and cerebrospinal fluid (CSF) samples with recoveries ranging from 96% to 108%.