Bacillus amyloliquefaciens EG025 was isolated from cheonggukjang, a traditional Korean fermented soybean paste. Among multiple isolates from diverse fermented foods, B. amyloliquefaciens EG025 uniquely exhibited potent gliadin-degrading activity, a trait with the potential to attenuate the immunogenicity of gluten peptides implicated in celiac disease. B. amyloliquefaciens EG025 maintained robust enzymatic activity across a wide pH spectrum (neutral to alkaline) and, remarkably, retained over 90.4% of its maximal activity following a 2-h exposure to pH 2. In addition to its gliadin-degrading capability, B. amyloliquefaciens EG025 demonstrated exceptional acid and bile tolerance, indicating a strong capacity to survive and function under the harsh conditions of the human gastrointestinal tract. Whole-genome sequencing and comprehensive annotation revealed biosynthetic gene clusters for secondary metabolites known to confer host benefits, alongside key probiotic functional genes involved in gliadin degradation, acid and bile resistance, stress response, mucosal adhesion, and de novo vitamin biosynthesis. Importantly, safety assessment confirmed the absence of concerning antibiotic resistance determinants or virulence factors, underscoring the strain's safety profile. Phylogenetic reconstruction further highlighted the genetic distinctiveness of EG025 relative to other B. amyloliquefaciens strains, emphasizing its novelty within the species. Collectively, these combined in vitro assay, genomic, and phylogenetic results underscore the significant probiotic potential of B. amyloliquefaciens EG025 and support its future development as a novel therapeutic candidate for improving gluten tolerance and managing celiac disease. Its safety and multifunctional attributes position it as a promising ingredient for functional foods targeted at gluten-sensitive populations.