2,6-Ditert-butylphenol (2,6-DTBP) is a major organic contaminant presenting in acrylic fiber manufacturing wastewaters.This compound is of high bio-resistance due to its complex structure which consists of one phenol group and two highly branched tert-butyl groups. This research attempted to improve the biodegradation efficiency of 2,6-DTBP through various strain immobilization methods. The stratified immobilization can settle oxygen transmission in the single microorganism immobilization, and can realize two-process reaction in the single device by choosing two symbiotic microorganisms. Two effective strains, named F-l-4 and F-3-4,which were screened out in our previous work, were used to degrade 2,6-DTBP after being immobilized in calcium alginate gel.Results indicate that the substrate removal efficiency of various immobilization methods follows the order: stratified ＞ single F-3-4 ＞mixed ≈single F-1-4. The immobilized biodegradation capacity was higher than the free one. After an incubation time of 12 d, 91%of 2,6-DTBP could be degraded by the stratified immobilization method, compared to 79% achieved by the mixed immobilization method with an initial 2,6-DTBP concentration of 100 mg/L. The stratified immobilization satisfies the oxygen demand nature of the aerobic F-3-4 and the facultative F-1-4, thus yielding the highest degradation efficiency. Both the outer layer strain F-3-4 and the inner layer strain F-1-4 can grow actively on the substrate of 2,6-DTBP, as illustrated by SEM images. This study shows that the highly bio-refractory compound, 2,6-DTBP, can be effectively degraded using appropriately immobilized microorganism strains.