The chiral recognition of enantiomers is a topic of great importance. In this study, a straightforward and effective chiral detection strategy was developed for distinguishing between tryptophan (TRP) enantiomers. The approach involved the preparation of a magnetic molecularly imprinted chitosan (MMIC) for the sample preparation, which was combined with a nitrocellulose membrane (paper-based analytical device, PAD) integrated with D-TRP covalently-grafted polymethacrylic acid (PAD_PMA_D-TRP). The discrimination between TRP enantiomers was achieved using AuNPs as a colorimetric probe. Indeed, the presence of D-TRP rapidly induced the aggregation of AuNPs due to its strong affinity to PAD-PMA_D-TRP, resulting in a noticeable color change of the AuNPs from red to purple. On the other hand, L-TRP did not induce any color change. The chiral analysis could be easily performed with the naked eye and/or a smartphone. The developed method exhibited a detection limit of 3.3 µM and it was successfully applied to detect D-TRP in serum samples, demonstrating good recovery rates. The proposed procedure is characterized by its simplicity, cost-effectiveness, rapidity, and ease of operation.