Sodium lignosulfonate reverse micelles (SLRMs) with vesicular structure were prepared by self-assembling in ethanol-water media and applied to encapsulate horseradish peroxidase (HRP). Results showed that sodium lignosulfonate (SL) could not form SLRMs until the ethanol content reached 63% when its initial concentration was 7.5 g L-1. Owing to strong electrostatic repulsion, solid spherical SLRMs gradually swelled to stable vesicular structures With an average size of 240 nm. The shell of the SLRM thickened when NaCI was added to screen the electrostatic interaction. HRP can be effectively encapsulated while retaining its activity in the hydrophilic core SLRM. When hydrogen peroxide was added to initiate the catalytic activity of HRP, SL molecules would be polymerized and the structure of SLRMs would be fixed. Furthermore, HRP immobilized in polymerized SLRMs showed high activity at a more acidic pH of 4 and at a lower optimal temperature decrease of 35 degrees C compared to free HRP. SLRM allows enzymes such as HRP to work at more acidic and lower temperature conditions.