In this work, a glassy carbon electrode (GCE) modified with large mesoporous carbon and Nafion composites (LMC/Nafion) was constructed and used for simultaneous determination of theophylline (TP) and caffeine (CF). The electrochemical behaviors of TP and CF on the LMC/Nafion/GCE were carefully investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The results show that the DPV peaks of the two analytes can be distinguished at the modified electrode with peak-to-peak separation about 150 mV, which means that LMC/Nafion/GCE is suitable for simultaneous determination of TP and CF. Under the optimized condition, the detection limits of TP and CF were 0.37 and 0.47 mu M (S/N = 3) with linear ranges of 0.8-180.0 mu M and 1.3-230.0 mu M, respectively. The proposed method was successfully applied to the simultaneous determination of TP and CF in human blood serum and beverages with reliable recovery. (C) 2013 Elsevier B.V. All rights reserved.