In this work, carbon dots (CDs) were produced by one-step ultrasound technology with glucose as the precursor. The effective synthesis of CDs was characterized by Raman spectroscopy and UV-visible spectra. The as-prepared CDs were fixed on a glassy carbon electrode (GCE) via electrochemical deposition to fabricate CDs/GCE, which simultaneously detected dopamine (DA), uric acid (UA), L-tryptophan (Trp) and theophylline (TP). With the differential pulse voltammetry technique, the oxidation peak currents of these four biomolecules were significantly enhanced on CDs/GCE compared to those on bare GCE. The potential differences of DA-UA, UA-Trp, and Trp-TP were computed as 145 mV, 381 mV, and 323 mV, respectively. The broad linear ranges were 0.5-50 mu M for DA, 3-75 mu M for UA, 1-65 mu M for Trp and 10-200 mu M for TP with limits of detection (LODs) of 0.007 mu M, 0.011 mu M, 0.11 mu M and 0.33 mu M (S/N = 3), respectively. The CD/GCE sensor had good stability and strong anti-interference ability and was applicable to detecting actual samples.