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[Display omitted] • An electrochemical aptasensor was designed to detect Acinetobacter baumannii in urine samples. • This aptasensor can detect Acinetobacter baumannii faster compared to the conventional method. • The stability of this aptasensor for 30 days was acceptable. Acinetobacter baumannii (A. baumannii) is a gram-negative bacterium that is responsible for causing a range of infections, including pneumonia, meningitis, and bloodstream infections and the detection of this bacterium is crucial for timely diagnosis and treatment of infections. The purpose of this work was to design and manufacture an electrochemical biosensor based on carbon nanofibers (CNFs), gold nanoparticles (AuNPs), and aptamer that could detect A. baumannii in urine samples. At first, polyacrylonitrile (PAN) polymer was electrospun and CNFs were formed during two stages of heat treatment including stabilization and carbonization, and Raman spectroscopy and X-ray diffraction (XRD) were used to characterize it. Then, these CNFs were used to modify the surface of the screen-printed carbon electrode (SPCE), and AuNPs were placed on the SPCE modified with CNFs using the electrodeposition method. Field emission scanning electron microscopy (FESEM) and energy dispersive X-Ray spectroscopy (EDS) were used to study the surface morphology of the modified electrode to determine the carbon and gold elements, respectively. Then the aptamer related to A. baumannii was placed on this modified electrode using a simple and cost-effective process and characterized by the cyclic voltammetry (CV) technique. Next, the spiked A. baumannii in urine samples was detected by this aptasensor in the range of 101 to 106 cfu mL−1 and limit of detection (LOD) to 0.681 cfu mL−1 with differential pulse voltammetry (DPV) technique. Salmonella typhimurium, Staphylococcus aureus and E. Coli were used to perform the selectivity test. In addition, its repeatability was checked and its stability for 30 days was more than 96 %. Finally, the results of this aptasensor were compared with the conventional method of bacterial culture, and this aptasensor was able to detect A. baumannii in a less time and with more sensitivity. [ABSTRACT FROM AUTHOR]