While the detection of tryptophan enantiomers is of great importance in the feld of pharmacy and biotechnology, signifcant limitations exist in their selective determination using conventional methods. A simple solution is to use immunochromatographic assays, thereby separating enantiomers and detecting them by electrochemical methods. In this study, the L-tryptophan enantiomer is captured by biotin-aptamer immobilized on an immunochromatographic test strip and recognized using a screen-printed electrode. PdCuCo/RGO nanocomposite is coated on the electrode surface in order to amplify the sensor sensitivity. The electrochemical measurements are performed by differential pulse voltammetry. At the optimal conditions, the sensor response shows a good linear relationship with the tryptophan concentration in the range of 0.08–20.0 μM, providing a detection limit of 0.03 μM. Since the sensor is stable and not susceptible to interfering species, it is possible to employ it as a reliable tool for the determination of the tryptophan level in biological samples and the diagnosis of a specifed disease.