Acinetobcter baumannii (A. baumannii) is responsible for various nosocomial infections, which is known as clinically important opportunistic pathogen. Therefore, rapid detection of this pathogen is critical to prevent the spread of infection and appropriate treatment [1]. Various methods have been developed for the detection of A. baumannii, such as Antibody-based assays, bacterial culture, nucleic acid-based assays, biochemical assays, and surface-enhanced Raman scattering [2]. Most of them are accurate but are accompanied by several problems in on-site detection, such as procedures being complicated and cumbersome, time is long, sensitivity is limited, highly sophisticated instruments and expensive, skilled manpower and the cost is high [2]. Therefore, to combat the aforementioned challenges, there is an urgent need for new powerful tools that are rapid and sensitive with the ability to detect A. baumannii on-site and that can guide the concerned person for taking appropriate control measures. Among various detection techniques, electrochemical methods have been recognized as one of the most promising technologies due to their high sensitivity, fabrication simplicity, accuracy, cost-effectiveness, selection, the possibility of mass production and ease of modification [3]. In the past few years, aptasensors were used for detecting the type of some bacteria [4]. In this work, an electrochemical aptasensor was developed for the ultrasensitive detection of A. baumannii by electrochemical impedance spectroscopy (EIS). The aptamer was linked to the surface of a carbon screen-printed electrode (CSPE) modified with the nanocomposite Fe3O4@SiO2@Gly for selective and label-free detection of A. baumannii by covalent bonding. The results of EIS studies showed that the constructed electrochemical aptasensor can specifically detect A. baumannii in the concentration range of from 1.0×103–1.0×108 CFU mL−1 and with a detection limit of 150 CFU mL−1 (S/N=3). In addition to its sens