This study investigated the immobilization of L-lactate oxidase (LOx) from Aerococcus viridans onto various porous carriers, including polymethacrylate, polyurethane, and agarose matrices functionalized with amine, Ni2+-loaded nitrilotriacetic acid (NiNTA), or epoxide groups. Both covalent attachment (via glutaraldehyde activation of amine groups or direct reaction with epoxide groups) and physical adsorption methods were evaluated. Key parameters assessed were carrier activity (Ac) and enzyme effectiveness (η) as a function of protein loading. Optimal results, achieving Ac up to 1450 U/g and η up to 65% (at low loading), were obtained with hydrophilic agarose carriers functionalized with amine groups, particularly when using glutaraldehyde activation for covalent binding. Effectiveness decreased sharply with increased protein loading, likely due to mass transfer limitations, especially for oxygen.