The encapsulation of probiotic cells in prebiotic polymers is one of the most effective techniques to maintain probiotic viability during gastrointestinal transit and storage. The purpose of this study was to fabricate a suitable colonic delivery system for Lactobacillus rhamnosus using alginate, pectin, and whey protein for oral administration. The effects of biopolymers at different concentrations and ratios under various sterilization temperatures on Lactobacillus rhamnosus viability were evaluated. The microbeads were evaluated for entrapment efficiency, shape and morphology by SEM. The viability of free and encapsulated Lactobacillus rhamnosus in simulated gastric fluids was also evaluated. Moreover, MTT assay was implemented to determine the cytotoxic effect of Lactobacillus rhamnosus microbeads using the colon cancer cell line Caco-2. Different signalling pathways in the Caco-2 cell line were also studied. The results showed that the formulae produced from polymers sterilized at low temperature had highest entrapment efficiency and viability for L. rhamnosus. The symbiotic effect of probiotics and prebiotics was observed mainly after incubation in simulated colon fluid for 4 h and 24 h. The mixture of components of the microbeads M12 showed anticancer effect on Caco-2 by upregulation of Bax and caspace-3 and downregulation of Bcl-2.