A novel bioaptasensing-based electrochemical method for determination of amifostine (AMF) is proposed. The electrochemical aptasensor is based on modification of a glassy carbon electrode with a nanocomposite consisting of silver nanoparticles @ MnFe Prussian blue analogue nanospheres (AgNPs@MnFePBA NS), followed by immobilization of aptamer via Ag-N bonds (aptamer/AgNPs@MnFePBA NS/GCE). Experimental parameters including pH, incubation time, and aptamer concentrations were optimized. Electrochemical impedance spectroscopy (EIS) and differential pulse voltammetric (DPV) techniques were utilized to quantify AMF. The anodic peak current (∆Ipa) and charge transfer resistance (∆Rct) differences increase in the presence of AMF. Under the optimal conditions, using the redox probe, the electrochemical aptasensor exhibited linear ranges of 0.34-45nmolL-1 and 0.69-45nmolL-1 with LODs of 0.11nmolL-1 and 0.23nmolL-1 for EIS and DPV, respectively. The aptasensor was used to determine AMF in human plasma and in the presence of interfering species with recoveries and RSDs in the range 97.8-103.2% and 2.2-4.2%, respectively. Graphical abstract.