Vitamin D3 (VitD3) deficiency is linked to numerous health conditions, increasing the demand for its bioavailable metabolite, 25-hydroxy-VitD3 (25OHVitD3). While chemical synthesis remains the standard production method, enzymatic approaches using steroid C25 dehydrogenases (S25DH) from Sterolibacterium denitrificans provide a highly selective, viable alternative. Herein, we present an S25DH-based bioelectrosynthesis approach, where optimizing electron transfer (ET) between S25DH and the working electrode is essential for achieving efficient bioelectrosynthesis. This study uses 8% antimony-doped tin oxide (ATO) thin-film electrodes to investigate S25DH immobilization dynamics, adsorption/desorption behavior, and electrocatalytic performance using in situ ATR-IR spectroelectrochemistry. Our findings indicate that S25DH binds to ATO surfaces via electrostatic and coordinative interactions, with surface-exposed histidine clusters playing a key role in stable immobilization..