The gut microbiota plays an important role in regulating brain function, influencing psychological and emotional stability. The correlations between conspecific aggression, gut microbiota, and physiological homeostasis were further studied in inbred laying chicken lines, 6₃ and 7₂, which were diversely selected for Marek’s disease, and they also behave differently in aggression. Ten sixty-week-old hens from each line were sampled for blood, brain, and cecal content. Neurotransmitters, cytokines, corticosterone, and heterophil/lymphocyte ratios were determined. Cecal microbiota compositions were determined by bacterial 16s rRNA sequencing, and functional predictions were performed. Our data showed that the central serotonin and tryptophan levels were higher in line 6₃ compared to line 7₂ (p < 0.05). Plasma corticosterone, heterophil/lymphocyte ratios, and central norepinephrine were lower in line 6₃ (p < 0.05). The level of tumor necrosis factor α tended to be higher in line 6₃. Faecalibacterium, Oscillibacter, Butyricicoccus, and Bacteriodes were enriched in line 6₃ birds, while Clostridiales vadin BB60, Alistipes, Mollicutes RF39 were dominated in line 7₂. From the predicted bacterial functional genes, the kynurenine pathway was upregulated in line 7₂. These results suggested a functional linkage of the line differences in serotonergic activity, stress response, innate immunity, and gut microbiota populations.