A highly selective electrochemical sensor was developed for the simultaneous determination of ascorbic acid (AA), dopamine (DA), uric acid (UA), tryptophan (Trp) and nitrite using a glassy carbon electrode (GCE) modified with an Au nanoparticle/TiO2 nanoparticle/carbon nanotube (Au/TiO2/CNT) composite. The Au/TiO2/CNT nanocomposite as a sensitive layer was used because it offers great electrocatalytic activity, large surface area, and abundant active sites. The Au/TiO2/CNT nanocomposite has good selectivity and remarkable sensitivity and could determine AA, DA, UA, Trp and nitrite. The Au/TiO2/CNT/GCE had linear response ranges for detecting AA, DA, UA, Trp and nitrite of 3−281, 0.3−15, 1−162, 1−162 and 4−225 μM, respectively. The detection limits were 1, 0.2, 0.6, 0.8 and 3 μM, respectively. In addition, a flexible sensing paper can be prepared via these nanocomposites for the simultaneous determination of these five species.