In this work, carbon nano-onions (CNOs) with particle sizes of 5−10 nm were prepared by the multi-potential step method. High-resolution transmission electron microscopy, infrared spectroscopy and Raman spectroscopy characterize the effective synthesis of CNOs. CNOs/GCEs were prepared by depositing the prepared CNOs onto glassy carbon electrodes (GCEs) by a drop-coating method. Examination of the electrocatalytic activity of the CNOs/GCE sensor by simultaneously detecting dopamine (DA), uric acid (UA), L-tryptophan (Trp) and theophylline (TP) using a differential pulse voltammetry technique. The results showed that the linear ranges of DA, UA, Trp and TP were DA 0.01−38.16 μM, UA 0.06−68.16 μM, Trp 1.00−108.25 μM, and TP 8.16−108.25 μM, and the detection limits (S/N = 3) were 0.0039 μM, 0.0087 μM, 0.18 μM and 0.35 μM, respectively. The CNOS/GCE sensor had good stability and could be used for the detection of actual samples.