Alzheimer's disease (AD) represents a slowly worsening neurodegenerative condition characterized by the accumulation of amyloid-β plaques and the formation of neurofibrillary tangles, ultimately impairing cognitive function. Aβ1–42 is the key isoform involved in its pathogenesis. Therefore, early and reliable detection of these peptides is essential to enable timely therapeutic intervention. In our work, we engineered an electrochemical biosensor by functionalizing a screen-printed gold electrode with 6-(ferrocenyl)hexanethiol, utilizing its reversible redox properties to provide more efficient electron transfer at the sensor's surface. The applicability of the aptasensor for quantifying Aβ1–42 was evaluated electrochemically by using square wave voltammetry (SWV). The method demonstrated an excellent linear correlation between the relative electrochemical response and Aβ1–42 concentration spanning 0.001–1000 pg/mL and the calculated LOD was 1.73 pg/mL. The sensor exhibited a high specificity against related biomolecules such as Aβ1–40, neurofilament light chain (NEFL), and TAU protein. Additionally, the biosensor exhibited acceptable performance in real biological matrices, including spiked serum and CSF, with minimal matrix interference. In comparison to traditional diagnostic techniques, this aptamer-based electrochemical platform provides a fast, cost-effective, and minimally invasive method for Aβ1–42 detection. These results demonstrate the potential of this biosensor for early AD diagnosis and point-of-care testing.