Salivary α-amylase (α-ALS) has drawn attention as a possible bioindicator for dental caries. Herein, combining the synergistic properties of multi-walled carbon nanotubes (MWCNTs), β-cyclodextrin (β-CD) and starch, an electrochemical sensor is constructed employing ferrocene (FCN) as an electrochemical indicator to oversee the progression of the enzymatic catalysis of α-ALS. The method involves a two-step chemical reaction sequence on a screen-printed carbon electrode (SPCE). X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, Field emission scanning electron microscope (FE-SEM), and Dynamic light scattering (DLS) were used to characterize the synthesized material, while Static water Contact angle measurements, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were performed to monitor each step of sensor fabrication. The electrochemical sensor permitted to detect α-ALS within the linear range of 0.5–280 U mL−1, revealing detection (LOD), and quantification (LOQ) values of 0.041 U mL−1, and 0.159 U mL−1, respectively. Remarkably, the sensor demonstrated exceptional specificity and selectivity, effectively discriminating against other interfering substances in saliva. Validation of the method involved analyzing α-ALS levels in artificial saliva with an accuracy range of 97 % to 103 %, as well as in real clinical saliva samples across various age groups.