Abstract: Whole body irradiation (WBI) injury is defined multi-organ damages caused by whole-body exposure to ionizing radiation. The traditional radioprotective drug, amifostine, has significant adverse effects. Probiotics are reported to have radioprotective function, although their therapeutic efficacy is low due to poor gastrointestinal tolerance and the insufficient retention and colonization in the colon. In this study, chitosan/tannic acid double-layer-coated Lactobacillus reuteri was prepared, which was encapsulated in calcium alginate hydrogel microspheres to get an engineered probiotic-loaded microsphere formulation. The bilayer coating was confirmed by twice inversions of zeta potentials. Moreover, the coating improved bacterial adhesion and aggregation. Optical microscopy revealed the smooth morphology of microspheres, laser confocal imaging showed the uniform distribution of coated bacteria in microspheres, and scanning electron microscopy exhibited pores in the surface. The microspheres exhibited in vitro gastrointestinal resistance with rapidly swelling in the colonic environment to release bacteria. All the animal experiments were approved by Academy of Military Medicine Sciences (Approval No: IACUC-DWZX-2024-P510) and conducted in compliance with relevant guidelines. The 6.5 Gy whole-body irradiated mouse model was established. Starting from 2 days prior to irradiation, probiotic-loaded microspheres were administered via oral gavage consecutively for 6 days. Compared with the model group, the bacteria-loaded microspheres demonstrated protective effects on the hematopoietic system by promoting the recovery of red blood cells and platelets, maintaining the morphology of splenic red pulp and white pulp, and preserving bone marrow nucleated cells along with their proliferative capacity. Engineered probiotics have expanded the spectrum of radioprotective drugs, offering novel insights for the development of live biotherapeutic products aimed at preventing and treating radiation-induced injury.