<p>We report a Ti₃C₂T_x MXene/MoS₂ heterostructure synthesised via in-situ hydrothermal growth to achieve structurally stabilized and high-performance non-enzymatic cholesterol sensing. Structural and chemical analysis confirmed the coexistence of oxygen-terminated Ti₃C₂T_x and 1T/1T′-MoS₂ with uniform elemental distribution across the heterostructure. Electrochemical characterization showed that the optimized Ti₃C₂T_x MXene/MoS₂ (1:1) heterostructure exhibited reduced charge-transfer resistance and an improved current response than the pristine materials. The optimized Ti₃C₂T_x MXene/MoS₂ (1:1) heterostructure-modified ITO-PET electrode enabled non-enzymatic cholesterol detection over a wide concentration range, exhibiting sensitivities of 268.6 µA µM^− 1 cm^− 2 (0.1 µM-1 µM) and 0.28 µA µM^− 1 cm^− 2 (1 µM-200 µM) for the low- and high-concentration regions, respectively, along with an excellent limit of detection of 25 nM. The electrode exhibited excellent selectivity, reproducibility, flexibility and 30 days stability. In addition, real sample analysis in the egg yolk showed a high recovery (94–97%) without any extraction treatment, validating the sensor’s applicability for biomedical and food-related monitoring. This study develops a robust material design framework for non-enzymatic biosensing and highlights the strong potential of Ti₃C₂T_x MXene/MoS₂ heterostructure for reliable cholesterol detection.</p> Graphical abstract <p></p>