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For the first time, a Ti3C2Tx–MXene and poly (3, 4-ethylenedioxythiophene): poly (styrenesulfonate) (PEDOT: PSS) composite-modified electrode has been developed for electrochemical detection of the bilirubin (BR) by molecularly imprinted ortho-phenylenediamine (o–PD). BR is a biomarker for liver-related diseases. High levels of BR imply liver dysfunction; hence, its exact and rapid measurement is indispensable to its immediate diagnosis and treatment. The synergistic effects of MXene and PEDOT: PSS not only enhanced the electrochemical conductivity and provided a large electroactive surface area for better MIP polymerization but also improved the sensitivity, stability, and electro-catalytic activity of the developed electrode. This is the first study to combine MXene/PEDOT: PSS and molecularly imprinted orthophenylenediamine for BR sensing, which individually have demonstrated potential, but whose combined effects have never been explored in the context of BR detection. The successful synthesis and deposition of composite is confirmed by field emission scanning electron microscopy (FESEM) along with energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). The electrochemical properties and surface morphology of the prepared electrode at every modification step were characterized by electrochemical techniques such as cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS), and FESEM respectively. The MXene/PEDOT: PSS composite as an electrode modifier exhibited sensing of BR in the clinical relevant range of BR in human serum 0.1–20 mg/dL with a detection limit of 0.002 mg/dL. Additionally, the prepared electrode has excellent reproducibility, stability, selectivity, and repeatability and also showed acceptable results for the sensing of BR in human serum sample. [ABSTRACT FROM AUTHOR]