[Background] The intestinal microflora of giant panda are abundant, and the population structure is related to the age of the host, living environment, seasonal changes and other factors, among which age is one of the important factors affecting the composition of intestinal microflora. [Objective] Study the diversity of Bacillus isolated from giant panda gut in different age, in order to explore the relationship between the diversity of Bacillus isolated from giant panda gut and age, which provide strain resources for the development of new probiotic. [Methods] Spread plate method was employed to isolate Bacillus. BOXA1R-PCR, 16S rRNA gene phylogenetic analysis and principal component analysis (PCA) were used to analyze the diversity of culturable Bacillus. Confrontation growth method and disk diffusion test were employed to test the antibacterial activity and antibiotic susceptibility of isolates. [Results] Total 90 strains belong to Bacillus genera were isolated. Based on the result of BOXA1R-PCR analysis, 41 representative strains were selected for further 16S rRNA gene sequencing. The result showed the isolates were affiliated to 6 species, including Bacillus subtilis, B. atrophaeus, B. velezensis, B. methylotrophicus, B. amyloliquefaciens, B. pumilus. The result of PCA showed the population composition of Bacillus in giant panda gut was influenced by age. All of representative isolates possessed CMCase ability, most of isolates exhibited antimicrobial activity to pathogens. All the 41 representative strains showed resistance to Penicillin G and susceptible to the other 9 antibiotics. [Conclusion] This study indicated that Bacillus isolated from giant panda gut was abundant and the distribution was influenced by age, which possessed great potential in developing probiotics and can be further explored.