Objective: The aim of this study was to explore the improvement of skin inflammation, behavioral characteristics, hormone levels， neurotransmitter levels and intestinal microbiota in a mouse model of 2,4-dinitrofluorobenzene (DNFB)-induced atopic dermatitis treated by eugenol combined with traditional Chinese medicine Wuxing music. Methods: A mouse AD model was established by DNFB induction for 21 consecutive days, during which the changes of dorsal skin lesions and the number of pruritus were recorded. On the 19th and 20th days, the open field experiment, the elevated 0 maze experiment, the suspension tail experiment and the forced swimming experiment were carried out. On the 21st day, the mice were sacrificed and dissected, and the organ index was calculated. At the same time, the levels of IgE, IL-4 and IL-10 in serum were determined by ELISA, the skin sections on the back were stained with HE and toluidine blue staining to observe the pathological changes of the skin, and the levels of corticosterone, dopamine and serotonin in the brain of mice were measured. In addition, 16S rRNA gene sequencing was used to analyze the composition of the intestinal microbiota in mice. Results: Compared with DNFB-induced AD mice, eugenol combined with traditional Chinese medicine Wuxing Music Therapy (EUG+MUSIC) group significantly reduced the skin lesion score and reduced the skin inflammatory cell infiltration. In behavioral tests, the EUG+MUSIC group showed more active locomotion, increased desire to explore unfamiliar environments, and increased struggling time in stressful situations. Neurotransmitter testing showed that the combination increased serotonin, dopamine, and corticosterone levels in the brains of mice. In addition, 16S rRNA gene sequencing results showed that DNFB-induced changes in the diversity of intestinal microbiota in AD mice, while EUG+MUSIC remodeled the gut microbial composition, with the relative abundance of certain gut microbes such as Ligilactobacillus and Lachnospiraceae increasing, while Parabacteroides and Bacteroides spp decreases. Spearman correlation analysis found a significant correlation between altered gut microbiota, AD-associated skin manifestations, and anxiety-depressive-like behaviors arising from comorbidities. Conclusion: Eugenol combined with the Five Elements Music Therapy of TCM has a positive effect on comorbid anxiety and depression-like behaviors by regulating the intestinal microbiota and improving the behavioral performance of AD mice, providing new ideas and methods for the improvement of AD and its anxiety-depressive behaviors.