The construction of a simple and convenient electrochemical platform for the recognition of enantiomers is crucial for the life sciences and medical fields. Herein, a portable electrochemical 3D platform based on a carbon paste electrode modified (CPE) with poly (alizarin red S) (PARS) and magnetite nanoparticles (Fe3O4NPs) for the recognition and determination of tryptophan enantiomers from whole blood samples was developed. Several methods, including scanning electron microscopy (SEM), differential pulse voltammetry (DPV), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS), were used to evaluate the morphologies and electrochemical behaviors of the modified electrode. The synthesized magnetite nanoparticles are studied using SEM with energy-dispersive X-ray analysis (EDAX). Under the optimum experimental conditions, the current intensity ratio of d-Trp to l-Trp (ID/IL) was found to be 1.32 at PARS/Fe3O4NPs/CPE, according to the results of differential pulse voltammetry. Furthermore, the developed platform showed a good linear correlation between response current intensity and tryptophan enantiomer concentration, ranging from 0.001 to 0.2 mM. The detection limits for both d-Trp and l-Trp were found to be 0.3 µM. This study demonstrates that the designed platform is capable of rapidly detecting the enantiomeric ratio in a non-racemic tryptophan mixed solution. Finally, the proposed platform was used to identify the Trp enantiomers in human whole blood, confirming the effectiveness of the modified electrode in analyzing real samples. The electrochemical 3D platform is thus used not only to recognize tryptophan enantiomers but also exhibits a significant promise for practical uses.