It is of great value to develop effective methods for accurately and simultaneously detecting ascorbic acid (AA) and dopamine (DA) in the field of biochemistry. This work reports a nonenzymatic electrochemical sensor for the simultaneous detection of AA and DA by employing a Co-modified MCM-41 (CoMCM-41) mesoporous molecular sieve as an efficient electrocatalytic material, which was synthesized by a two-step hydrothermal method. Subsequently, the high structural organization of the CoMCM-41 mesoporous structure was characterized, and the electrocatalytic performance of CoMCM-41 toward AA and DA oxidation was then evidenced by the catalytic effect of different electrodes modified with or without CoMCM-41. By virtue of the superior electrocatalytic activity of the CoMCM-41, a much wider peak potential difference (ΔEpa) of 310 mV was obtained for the oxidation of AA and DA in their mixture solution, and the parameters that influenced the electrochemical signals of the modified electrode were also optimized. Under optimal conditions, a good linear response to AA and DA was observed on the CoMCM-41 modified electrode. For individual detection of AA and DA, the linear ranges were 7 ~ 105 μM and 5 ~ 110 μM respectively, while the linear response range was 20 ~ 100 μM for simultaneous detection of AA and DA. Satisfactory recovery results were obtained when the fabricated sensor was applied to determine AA in orange juice and DA in madopar pill samples.