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Microencapsulations of Lactobacillus acidophilus in xanthan-chitosan (XC) and xanthan-chitosan-xanthan (XCX) polyelectrolyte complex (PEC) gels were prepared in this study. The process of encapsulation was optimized with the aid of response surface methodology (RSM). The optimum condition was chitosan of 0.68%, xanthan of 0.76%, xanthan-L. acidophilus mixture (XLM)/chitosan of 1:2.56 corresponding to a high viable count (1.31 ± 0.14) × 1010 CFU·g−1, and encapsulation yield 86 ± 0.99%, respectively. Additionally, the application of a new encapsulation system (XC and XCX) in yoghurt achieved great success in bacterial survival during the storage of 21 d at 4 °C and 25 °C, respectively. Specially, pH and acidity in yogurt were significantly influenced by the new encapsulation system in comparison to free suspension during 21 d storage. Our study provided a potential encapsulation system for probiotic application in dairy product which paving a new way for functional food development.