<p>Regarding the importance of early diagnosis of ovarian cancer and its progression monitoring, an ultrasensitive sandwich-type electrochemical immunosensor was developed to detect cancer antigen 125 (CA 125) biomarker. A hybrid nanocomposite was synthesized by integrating COOH-functionalized multi-walled carbon nanotubes (MWCNT-COOH) with a trimetallic NiYFe layered double hydroxide (NiYFe-LDH). To further improve the electrochemical activity, gold nanoparticles (AuNPs) were deposited onto the resulting nanohybrid structure. The final Au@LDH/MWCNT nanocomposite was characterized using standard methods and subsequently employed to modify screen-printed carbon electrodes (SPCEs) for capturing the sandwich structure of the immunosensor. The high specific surface area and biocompatibility of the nanocomposite provided an appropriate microenvironment for the effective capture of primary antibodies (Ab1) and preserving the integrity of the sandwich structure during signal analysis. The output response of the immunosensor was measured utilizing differential pulse voltammetry (DPV), revealing a wide linear range of 0.1 U mL^− 1 to 600 U mL^− 1 and a low detection limit of 0.0013 U mL^− 1. The immunosensor demonstrated excellent selectivity, stability, and reproducibility. Furthermore, its reliable performance for the clinical applications was confirmed by evaluating recoveries using the standard addition and the standard enzyme-linked immunosorbent assay (ELISA) methods. Additionally, the results exhibited that the proposed platform can be further developed for the detection of other cancer-related biomarkers.</p> Graphical Abstract <p></p>