An yttrium hexacyanoferrate nanoparticle/multi-walled carbon nanotube/Nafion (YHCFNP/MWNT/Nafion)-modified glassy carbon electrode (GCE) was constructed. Several techniques, including infrared spectroscopy, energy dispersive spectrometry, scanning electron microscopy and electrochemistry, were performed to characterize the yttrium hexacyanoferrate nanoparticles. The electrochemical behavior of the YHCFNP/MWNT/Nafion-modified GCE in response to l-cysteine oxidation was studied. The response current of l-cysteine oxidation at the YHCFNP/MWNT/Nafion-modified GCE was obviously higher than that at the bare GCE or other modified GCE. The effects of pH, scan rate and interference on the response to l-cysteine oxidation were investigated. In addition, on the basis of these findings, a determination of l-cysteine at the YHCFNP/MWNT/Nafion-modified GCE was carried out. Under the optimum experimental conditions, the electrochemical response to l-cysteine at the YHCFNP/MWNT/Nafion-modified GCE was fast (within 4s). Linear calibration plots were obtained over the range of 0.20–11.4μmolL−1 with a low detection limit of 0.16μmolL−1. The YHCFNP/MWNT/Nafion-modified GCE exhibited several advantages, such as high stability and good resistance against interference by ascorbic acid and other oxidizable amino acids.