Accurate measurement of tryptophan (Trp) levels is crucial for clinical and research purposes, such as nutritional assessment, disorder diagnosis, condition management, and the study of the role of Trp in disease pathophysiology. Herein, the intercalation of 1-octyl-3-methylimidazolium chloride [OMIM]⁺Cl⁻ ionic liquids (ILs) between the layers of Ti₃C₂ MXenes results in a 3D porous structure with a large active surface area and high interlayer spacing (d-spacing). Confined [OMIM]⁺ ions enhance the electroactive sites and Trp transfer pathways at the Ti₃C₂ MXene and IL interfaces and improve the electron transfer efficiency for Trp oxidation, improving Ti₃C₂ MXene stability via strong π‒π and electrostatic Ti₃C₂ MXene‒IL interactions. Under optimal conditions, the sensor demonstrated a broad detection range for Trp, ranging from 0.001 to 240 µM, with a low limit of detection of 0.06 nM (S/N = 3). Owing to its exceptional stability, selectivity, and reproducibility, the proposed IL-Ti₃C₂/GCE exhibited significant potential for detecting Trp in real amino acid granules and urine samples.