Abstract: This study aimed to immobilize D-allulose 3-epimerase from Ruminococcus sp. (RDAE) using cross-linked enzyme aggregates (CLEAs) technology. The immobilized enzyme (RDAE-CLEA) was prepared by optimizing the precipitant polyethylene glycol (PEG4000) concentration, cross-linker dextran aldehyde concentration, and cross-linking time. The enzymatic properties of the free enzyme (RDAE) and the immobilized enzyme (RDAE-CLEAs) were comparatively evaluated. Furthermore, the structural changes in RDAE before and after immobilization were analyzed using scanning electron microscopy, infrared spectroscopy, and molecular dynamics simulations. In the absence of stabilizers, the RDAE-CLEAs prepared with 36% (w/v) PEG4000 and 2.73 mg/mL dextran aldehyde at 4 h achieved an enzyme activity recovery rate of 55.88%. Compared with the free enzyme, the immobilized enzyme exhibited siginificantly improved environmental tolerance (P<0.05) at pH 6.0~7.5 and 30~60 ℃, retaining 59.77% initial activity after 10 reuse cycles. Structural analysis revealed that RDAE underwent significant conformational changes during cross-linking, and the binding tightness between subunits in the enzyme tetramer was reduced. The study developed RDAE-CLEAs with good reusability and elucidated the dual role of surface lysine residues in both facilitating covalent cross-linking and maintaining the rigidity of the tetrameric structure of RDAE. Considering the critical impact of tetramer rigidity on the thermal stability of RDAE, developing novel cross-linking strategies that preserve lysine residues on the enzyme surface would substantially improve the application potential of RDAE-CLEAs.