The objective of this study was to develop a novel immobilized system using kefir lactic acid bacteria and sugar cane pieces for the production of fermented milk. Lactobacillus kefiranofaciens HL1, Lactobacillus kefiri HL2, Leuconostoc mesenteroides HL3, and Lactococcus lactis HL4 were isolated from Taiwanese kefir grains and immobilized on pieces of sugar cane using adsorption. Scanning electron micrographs of the cell-immobilized sugar cane pieces (CISCP) showed that the microorganisms were embedded within the porous structures of the sugar cane pieces. During 28 cycles of repeated batch fermentation, viable cells on both sugar cane pieces and fermented products were maintained at 10 log cfu/g (cfu/mL). Random amplified polymorphic DNA PCR analysis revealed that Leu. mesenteroides HL3 (29-43%) and Lc. lactis HL4 (31-49%) were predominant on the CISCP, and the fermented samples had 79% Lc. lactis HL4. When tracking fermentation parameters, the data on the microbial, chemical, and physical properties of the fermented milk suggested that the CISCP had stable fermentative ability over the course of successive fermentations. We found an enhancement of the acid-producing ability of CISCP as the number of fermentations increased, with a significant growth in titratable acidity from 0.65 to 0.81% by the end. Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.