Probiotics have been proven to play a positive role in the prevention and treatment of diabetes. The gut microbiota of different populations harbors an extremely rich diversity of species and strains. This study aimed to explore potential new probiotic resources from the intestine of Tajik children with the goal of discovering new strains that can be used for the prevention and treatment of diabetes. In this study, Bifidobacterium strains were isolated from the intestine of healthy Tajik children in Xinjiang and identified using groEL gene sequencing. These strains were then screened for their in vitro probiotic characteristics to identify potential anti-diabetic strains. The results showed that 1579 Bifidobacterium strains were isolated and purified from the intestine of 62 children, with Bifidobacterium longum subsp. longum being the dominant ones (591 isolates). A total of 76 sequenced strains of B. longum subsp. longum from 33 samples containing B. longum, at least one strain of which was from each sample, were analyzed for in vitro probiotic characteristics. The results showed that five B. longum subsp. longum strains (TXX165-6, TXX127-13, TXX13-17, TXX51-1, and TXY63-27) exhibited a survival rate exceeding 60% in simulated gastrointestinal fluid and demonstrated good carbon source utilization capabilities. All of their cell-free supernatants exhibited strong antioxidant activity and α-glucosidase inhibitory activity, scavenging over 82% of 2,2’-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) cation radical and over 68% of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. Strain TXX165-6 exhibited the highest surface hydrophobicity (78.46%) and surface self-aggregation ability (49.15%). Strain TXX51-1 displayed the second-highest surface hydrophobicity (67.30%) and inhibited 68.75% of α-glucosidase activity, which was significantly superior to the model probiotic Lacticaseibacillus rhamnosus GG (20.15%). Therefore, strains TXX165-6 and TXX51-1 are promising candidates for further animal experiments to assess their potential as potential anti-diabetic strains.