This study reports the simple, selective and sensitive voltammetric detection of riboflavin (RF) using methylene blue (MB) incorporated sulfonic acid functionalized mesoporous silica microspheres (MSM), represented as MB-SO₃H-MSM. MB-SO₃H-MSM is synthesized and characterized by spectroscopic and microscopic methods. This material is coated on a glassy carbon (GC) electrode (symbolized as GC/MB-SO₃H-MSM) to utilize it in electroanalytical applications. The electrochemical behavior of MB-SO₃H-MSM is established using the GC/MB-SO₃H-MSM electrode by cyclic voltammetry (CV) and electrochemical impedance spectroscopy techniques. The electrochemical behavior of RF at the GC/MB-SO₃H-MSM electrode is also studied by CV. Compared to bare GC and SO₃H-MSM coated GC, the GC/MB-SO₃H-MSM electrode shows favorable electron transfer kinetics as well as an enhanced and stable electrochemical response of RF. Furthermore CV and differential pulse voltammetry (DPV) are used for the quantitative determination of RF at the GC/MB-SO₃H-MSM electrode. The DPV response shows two linear calibration ranges of 10.0 nM to 15.0 μM and 15.0 to 50.0 μM. The detection limit based on the first linear calibration range is calculated as 5.0 nM with a sensitivity of 393.0 μA mM⁻¹ cm⁻². The fabricated sensing scaffold shows an excellent selectivity for RF over other soluble vitamins and interfering ions. The stability, reproducibility and determination of RF in pharmaceutical products are also demonstrated effectively.