Bilirubin is the yellow-orange bile pigment found in blood, mostly bound to the plasma protein albumin. Bilirubin is an important serum biomarker used in clinical medicine for assessing hemolysis, hepatic function and cardiovascular risk [1]. This molecule originates from the degradation of the heme moiety in hemoglobin, other hemoproteins, such as cytochromes, catalase, peroxidase and tryptophan pyrrolase, and free heme. Higher and lower concentrations of bilirubin are associated with certain diseases. For instance, jaundice, caused by high bilirubin levels in the blood, is associated with gallbladder and liver diseases (e.g., cirrhosis, hepatitis), blood infection, transfusion reaction or haemolytic diseases of the newborn (cell destruction). Conversely, low levels of bilirubin are associated with anaemia and coronary artery diseases [2]. Therefore, to know the treatment and study of the mechanism of these disease determinations of bilirubin in biological fluids can be of analytical interest. Among many techniques, electrochemical techniques as alternative methods have also received much interest due to their higher selectivity, faster and simple operation, lower cost, quick response, and therefore, have become of considerable importance for determination of analytes. Carbon electrodes such as glassy carbon electrode (GCE) and carbon paste electrode (CPE) possess many advantages including low fabrication cost, wide accessible potential window, chemical and electrochemical stability so that, in many cases, these electrodes are superior with respect to other electrodes [3]. In order to improve their electrochemical performance, glassy carbon electrodes have been modified with nanosized materials. In this paper, MXene/ZnO nanocomposite based electrochemical sensor was fabricated for the determination of bilirubin. Electrochemical studies were carried out by using cyclic voltammetry (CV), Differential pulse voltammetry (DPV) and chronoamperometry (CHA). The DPV data showed that the obtained anodic peak currents were linearly dependent on the bilirubin concentrations in the range of 0.1–600.0 μM, with the detection limit of 0.05 μM. The prepared electrode was successfully applied for the determination of bilirubin in real samples. Moreover, the modified electrode exhibited excellent stability and reproducibility.