The objective of this research was to encapsulate probiotics by alginate hydrogel beads based on an in situ cultivation method and investigate the influences on the cell loading capacity, surface and internal structure of hydrogel beads and in vitro gastrointestinal digestion property of cells. Hydrogel beads were prepared by extrusion and cultured in MRS broth to allow probiotics to grow inside. Up to 10.34 ± 0.02 Log CFU/g of viable cell concentration was obtained after 24 h of in situ cultivation, which broke through the bottleneck of low viable cell counts in the traditional extrusion method. Morphology and rheological analyses showed that the structure of the eventually formed probiotic hydrogel beads can be loosed by the existence of hydrogen bond interaction with water molecules and the internal growth of probiotic microcolonies, while it can be tightened by the acids metabolized by the probiotic bacteria during cultivation. In vitro gastrointestinal digestion analysis showed that great improvement with only 1.09 Log CFU/g of loss in viable cells was found after the entire 6 h of digestion. In conclusion, the current study demonstrated that probiotic microcapsules fabricated by in situ cultivation method have the advantages of both high loading capacity of encapsulated viable cells and good protection during gastrointestinal digestion.