In this work, physical adsorption was used for immobilization of proline dehydrogenase onto a magnetic mesoporous silica nanomaterial. The immobilization and electrocatalytical activity of proline dehydrogenase entrapped in a magnetic mesoporous silica nanomaterial was studied using cyclic voltammetry, differential pulse voltammetry, and square wave voltammetry. The magnetic mesoporous silica networks having a high surface area (362m2g-1) exhibited excellent properties for entrapment of proline dehydrogenase. The applied approach led to better resistance to temperature and pH inactivation in comparison to the free enzyme. The electrocatalytic current response of proline dehydrogenase entrapped in a magnetic mesoporous silica nanomaterial toward oxidation of L-proline was maintained in the analytical solution temperature up to 70°C. The entrapped proline dehydrogenase was casted onto a polycysteine-modified glassy carbon electrode. The electrode was evaluated as an electrochemical biosensor for electrooxidation and determination of L-proline in phosphate buffer solution. A cyclic voltammetry study indicated that the oxidation process of proline is irreversible and is diffusion controlled. The electrochemical behavior was further exploited as a sensitive detection scheme for L-proline determination by differential-pulse voltammetry. Under optimized conditions, the concentration range and detection limit were 0.01-0.15μM and 0.006μM, respectively. The method was applied to the assay of L-proline in whole blood and normal and malignant cell line lysates (normal cell (L929); gastric cancer cell-CAT 3, colon cancer cell-HCT, colon cancer cell-SW, and breast cancer cell-MCF7).