An imbalance of l-tryptophan (l-Trp), a basic component of a healthy diet, is harmful to human health. Traditional methods for detecting l-Trp have many limitations. To correct a deficiency or excess of l-Trp in human diets, it is necessary to develop a novel method that is rapid, low-cost, and high-sensitivity. Herein, a molecularly imprinted polysaccharide electrochemical sensor termed MIP/CS/MWCNTs/GCE (molecularly imprinted polymer/chitosan/multiwalled carbon nanotubes/glassy carbon electrode) targeting l-Trp was first constructed on a glassy carbon electrode, which was modified with multiwalled carbon nanotubes and chitosan using bifunctional monomers. The MIP/CS/MWCNTs/GCE obtained a wide linear range (1-300 μM) for detecting l-Trp and accurately detected the proportion of l-Trp in mixtures of Trp enantiomers. In milk samples, the spiked recoveries of l-Trp were 86.50 to 99.65%. The MIP/CS/MWCNTs/GCE electrochemical sensor possessed good recognition and detection performance for l-Trp and has promising potential for practical application.