We demonstrate label-free electrochemical impedance spectroscopy (EIS) based alkaline phosphatase (ALP) detection using gold nanoparticles (AuNPs), electrochemically engineered Au-nano-Dendroids, and graphene oxide (GO) nanocomposite. These nanomaterials were sequentially deposited onto the screen-printed carbon electrode (SPCE) and antibodies against ALP (anti-ALP) were immobilised using a carbodiimide bioconjugation process. The sensor probe has been characterised extensively using TEM, EDX, SAED, XRD, FE-SEM, FTIR, DIC and electrochemical techniques. The analytical performance of the fabricated biosensor was evaluated using EIS, where the linear dynamic range and limit of detection were 100–1000 U/L and 9.10 (±0.12) U/L respectively. The developed biosensor showed high selectivity towards ALP with negligible interference (ksel ≪ 1; n = 3) from coexisting molecules. The sensor probe recovered ALP between 108.84% and 172.50% (n = 3) in human serum samples, and was used to estimate ALP in clinical serum samples where the level was found to be 83.15 U/L — comparable with the standard technique used in hospitals. Shelf life, stability and reproducibility have also been evaluated.