D-Amino acid oxidase (DAAO) presents a paragon for effective use of biocatalytic O2-dependent transformations in fine-chemical and pharmaceutical synthesis at the industrial scale. Solid-supported DAAO immobilizates are applied to continuous processing, but their activity and stability are often inadequate. Targeted immobilization development is restricted by insufficient knowledge of physical and biochemical factors governing the performance of heterogeneous DAAO catalysts. We have applied real-time optical sensing of the O2 availability in luminescence-labeled porous Sepabeads and ReliSorb carriers to quantify diffusional restrictions in DAAO immobilizates differing in the mode of enzyme attachment to the solid surface. We show that noncovalent oriented immobilization of DAAO (from Trigonopsis variabilis) resulted in high retention of the original enzyme activity (≥60 %), whereas covalent multipoint fixation caused massive (up to 90 %) activity loss. Depletion of O2 inside the solid immobilizates became limiting for enzyme catalytic effectiveness at activity loadings as low as 5 units gcarrier−1. Slow pore diffusion was principally responsible for the observed large mass-transfer resistance, and this provides a main starting point for process intensification.