Acinetobacter baumannii (A. baumannii) is responsible for various nosocomial infections, which is known as a clinically crucial opportunistic pathogen. Therefore, rapid detection of this pathogen is critical to prevent the spread of infection and appropriate treatment. Biological detection probes, such as aptamers and synthetic receptors can be used as diagnostic layers to detect bacteria. In this work, an electrochemical aptasensor was developed for the ultrasensitive detection of A. baumannii by electrochemical impedance spectroscopy (EIS). The aptamer was immobilized on the surface of a CSPE modified with the nanocomposite Fe3O4@SiO2@Glyoxal (Gly) for selective and label-free detection of A. baumannii. The charge transfers resistance (Rct) between redox couple [Fe(CN)63−/4-] and the surface of aptasensor in the Nyquist plot of EIS study was used as electroanalytical signal for detection and determination of A. baumannii. The obtained results showed that the constructed aptasensor could specifically detect A. baumannii in the concentration range from 1.0 × 103–1.0 × 108 Colony-forming unit (CFU)/mL and with a detection limit of 150 CFU/mL (S/N = 3). In addition to its sensitivity, the biosensor exhibits high selectivity over some other pathogens. Therefore, a simple, inexpensive, rapid, label-free, selective, and sensitive electrochemical aptasensor was developed to detect A. baumannii.