The aim of this study was to investigate the impact of jackfruit inner skin fibre (JS) incorporated with whey protein isolate (WPI) and soybean oil (SO) as a wall material for probiotic encapsulation to improve probiotic stability against freeze-drying and gastrointestinal (GI) tract conditions.Bifidobacterium bifidumTISTR2129,Bifidobacterium breveTISTR2130, andLactobacillus acidophilusTISTR1338 were studied in terms of SCFA production and the antibiotic-resistant profile and in an antagonistic assay to select suitable strains for preparing a probiotic cocktail, which was then encapsulated. The results revealed thatB. breveandL. acidophiluscan be used effectively as core materials. JS showed the most influential effect on protecting probiotics from freeze-drying. WPI:SO:JS at a ratio of 3.9:2.4:3.7 was the optimized wall material, which provided an ideal formulation with 83.1 ± 6.1% encapsulation efficiency. This formulation presented > 50% probiotic survival after exposure to gastro-intestinal tract conditions. Up to 77.8 ± 0.1% of the encapsulated probiotics survived after 8 weeks of storage at refrigeration temperature. This study highlights a process and formulation to encapsulate probiotics for use as food supplements that could provide benefits to human health as well as an alternative approach to reduce agricultural waste by increasing the value of jackfruit inner skin.