Tryptophan levels in the human body are closely related to disease development and metabolic processes, but identification and quantification of tryptophan enantiomers at the single-cell level is still very challenging now. Herein, the mono-(6-ethanediamine-6-deoxy)-β-cyclodextrin (β-CD)-modified carbon nanopipet (CNP) was fabricated, and high-enantioselective electrochemical detection of tryptophan was achieved. Interestingly, the selectivity of the prepared CNP toward l-tryptophan (l-Trp) and d-Tryptophan (d-Trp) could be modulated by adjusting the solution pH. Moreover, besides measuring the l-Trp concentration in a preserved cell environment, the fabricated tip could also be used to monitor the dynamics of l-Trp metabolism in the presence of representative amino acids. This work would offer a new approach to measure Trp enantiomers and reveal tryptophan metabolic pathways at the single-cell level.