We developed a site-directed immobilization strategy exploiting the competitive inhibitor Remazol Brilliant Blue R (RB) to orient horseradish peroxidase (HRP) on functionalized glass surfaces. RB was covalently bound to poly(ethylene-alt-maleic anhydride) (PEMA)-coated substrates, enabling HRP binding via its active site. Subsequent contact with a second PEMA-functionalized surface facilitated covalent immobilization of HRP with controlled orientation. Interfacial layer assembly and the comparative random control were characterized by AFM and XPS analyses, while successful RB acetylation was confirmed by NMR and ATR-IR spectroscopy. Directed contact approaches using stepwise RB and PEMA+RB functionalization yielded HRP loadings with specific activities approximately twofold higher than non-directed immobilization. Our inhibitor-mediated method preserves the native enzyme structure without genetic modification, offering a straightforward platform for enhancing enzyme performance. This strategy is promising for applications in biosensors, biocatalysis, and enzyme-based devices requiring efficient enzyme immobilization.