Objective To analyze the relationship between intestinal floras and serum interferon-γ (IFN-γ), interleukin-4 (IL-4) levels in children with Mycoplasma pneumoniae pneumonia (MPP). Methods A total of 106 MPP children admitted to the hospital from October 2016 to October 2020 were enrolled as the research objects. According to different courses of disease, they were divided into acute phase group (n=46) and recovery phase group (n=60). Fifty healthy children undergoing physical examination during the same period were enrolled as control group. The counts of intestinal floras (Bifidobacterium, Eubacterium, Lactobacillus, Escherichia coli, Peptococcus), serum IFN-γ and IL-4 levels were compared among the three groups. The correlation between the intestinal floras and serum IFN-γ, IL-4 levels was analyzed. Results The counts of Bifidobacterium, Eubacterium, Lactobacillus, Escherichia coli and Peptococcus in acute and recovery phase groups were lower than those in control group, which were lower in acute phase group than recovery phase group (P＜0.05). The levels of serum IFN-γ and IL-4 in acute and recovery phase groups were higher than those in control group, which were higher in acute phase group than recovery phase group (P＜0.05). Spearman correlation analysis showed that counts of Bifidobacterium, Eubacterium, Lactobacillus, Escherichia coli and Peptococcus were negatively correlated with serum IFN-γ and IL-4 levels (P＜0.05). Conclusion The number of intestinal floras is little, while levels of serum IFN-γ and IL-4 are high in MPP children. The levels of serum IFN-γ and IL-4 increase with the decrease of intestinal floras.