Multilayer microencapsulation has been used as an alternative to improve the stability of bioactive compounds submitted to processing and storage conditions. Thus, this work aimed to produce and characterize calcium alginate particles with Lactobacillus acidophilus and coat them with different multilayers of milk whey protein and sodium alginate through electrostatic interactions. Particles were obtained by ionic gelification, using a double fluid spray nozzle. Microorganism resistance effects with the number of layers was assessed, as well as the morphology and mean size of the coated particles. Survival of free and encapsulated microorganisms during the simulation of gastrointestinal tract passage, thermal treatments, and feasibility after storage at different times and temperature were also assessed. Encapsulation efficiency was greater than 80% in all treatments, while diameter ranged from 107 μm to 222 μm. Microencapsulated Lactobacillus acidophilus showed feasibility during the simulated gastrointestinal conditions, wherein the multilayers of the particle with milk whey protein (A1) provided the greatest protection for the probiotic microorganisms allowing for microorganism release in different parts of the tract (9.19 CFU g-1). In the thermal resistance tests, multilayered particles showed better feasibility compared with free cells that could not resist all temperatures tested, whereas A1 treatment showed better results for up to 120 minutes at 63⁰C (8.82 CFU g -1). Storage for up to 120 days at refrigeration and freezing temperatures were more efficient for all types of microparticles when compared to free cells. Results found in this research suggest that microparticles multilayered by electrostatic interaction present an effective method to improve probiotic feasibility during storage, thermal treatments, and under gastrointestinal conditions.