The aim of our research was to prepare probiotic microcapsules with alginate, starch, glycerol and to evaluate effect of different microcapsules formula on their properties. Lactobacillus acidophilus ATCC 4356 were encapsulated in alginate microcapsules by phase separation coacervation method. The mixture of 3% sodium alginate, 5% or 10% starch, 15% glycerol and cell biomass were dripped into 2% calcium chloride solution (all concentration were in % w/v) and the newly formed microcapsules were incubated for approximately 30 mins in order to obtain the rigid structure. After freeze-drying of the microcapsules, the more spherical shape and higher bacteria density were observed with the formula of alginatestarch-glycerol in compare to the formula without glycerol. The images from the scanning electron microscope (SEM) of cross section of those microcapsules with glycerol showed a uniform concentric circle inner layers, which was not seen in the formula without glycerol. Both starch and glycerol improved the survival of L. acidophilus after freeze-drying process and help to maintain low moisture of obtained microcapsules. After 3 months storage at 4°C, the viability ofL. acidophilus in both types of microcapsules decreased but remained at 108 (cfu/g) colonies forming unit per gram. The highest bacteria density of 1.91×109 cfu/g were observed with the formula of alginate-glycerol-10% starch as well as the virtually constant low water activity (0.011).