Background: This study aims to investigate whether immune dysregulation and gut microbiome alteration are exacerbated in atopic dermatitis (AD) with food allergy (ADFA) and potential treatment strategies. Methods: Total 159 children with AD (tAD) were divided into two groups: AD without-food allergy (ADNFA) and with food allergy (ADFA); 100 children without AD were included as control. Eosinophil counts and total serum IgE levels were measured by routine methods, serum food-specific IgE levels by quantitative fluorescence immunoassay, and serum cytokine levels by multi-microsphere flow immunofluorescence. The intestinal microbiota was evaluated in fecal specimens using metagenomic sequencing. A novel ADFA mouse model was generated to evaluate whether probiotic candidates identified from human fecal samples contributed to the improvement in ADFA pathology. Results: The levels of eosinophils, IgE, IL-2, TNF-α, IL-4, IL-5, IL-6, IL-10, IL-17, IL-12P70 and IFN-α were elevated in tAD compared to normal controls. Compared with ADNFA, the levels of eosinophils, IgE and IL-5 were persistently increased, while IFN-γ was decreased, the species of Lactococcus lactis (L. lactis) was reduced in ADFA. Compared with AD, the ADFA model had more severe skin lesions on the back and significantly higher serum OVA-specific IgE, IL-4 and IL-5. Following oral administration of L. lactis ( L. lactis 1.1936+1.3992), skin lesions in ADFA mice was significantly improved. The levels of OVA-specific IgE, IL-4 and IL-5 decreased in a dose-dependent manner. Conclusions: Food allergy aggravates immune dysregulation and gut microbiome dysbiosis in children with AD. L. lactis could be a candidate probiotic for the treatment of ADFA.