The rising prevalence of autism spectrum disorder (ASD) significantly impacts children's development. Emerging evidence suggests that modulating gut microbiota may alleviate neurodevelopmental disorders, including ASD. Probiotics represent a promising approach for targeting gut microbiota in ASD management due to their safety profile and absence of side effects. This study evaluated a 12-week intervention with Bifidobacterium longum subsp. longum CCFM1077 in children with ASD. The probiotic group showed significant reductions in childhood autism rating scale (CARS) scores with notable improvements in affect and anxiety reactions. 16S rRNA sequencing revealed that the intervention significantly increased the relative abundances of Bifidobacterium, Lactobacillus, and Roseburia, which were initially depleted in these children. Concurrent with symptom improvement, serum 25-hydroxyvitamin D (25OHD) levels significantly increased in the probiotic group. In contrast, the placebo group exhibited no significant symptom alleviation, gut microbiota modulation, or changes in serum 25OHD levels. These findings highlight the potential of probiotic intervention as a food science-based adjunctive therapeutic approach for ASD in clinical practice.