Idiopathic macular holes are common ophthalmic manifestations with unknown pathogenesis. Thus far, there has been minimal research regarding the causes of idiopathic macular holes, especially with respect to the underlying immune mechanism. To provide clarity regarding the treatment and prognosis of idiopathic macular holes, specifically regarding the levels of cytokines in affected patients, this study examined and analyzed multiple cytokine levels in aqueous humor from patients with idiopathic macular holes.This comparative cross-sectional study included 38 patients in two groups: a cataract control group (n = 17) and an idiopathic macular hole group (n = 21). The levels of 48 cytokines in aqueous humor were detected by multiplex analysis with antibody-coupled magnetic beads. The Kolmogorov-Smirnov test was used to check whether the data were normally distributed; Student's t-test and the Mann-Whitney U test were used to assess differences in cytokine levels between the two groups. Spearman correlation analysis was used to assess relationships among cytokine levels in the experimental group. Signaling pathways containing cytokines with significantly different expression in the experimental group were identified.There were significant differences in aqueous humor cytokine levels between patients with idiopathic macular holes and patients in the cataract control group. Notably, hepatocyte growth factor (p = 0.0001), GM-CSF (p = 0.0111), and IFN-γ (p = 0.0120) were significantly upregulated in the experimental group, while TNF-α (p = 0.0032), GRO-α (p < 0.0001), and MIF (p < 0.0001) were significantly downregulated in the experimental group. Furthermore, the GM-CSF level showed significant positive correlations with levels of IL-1 (r = 0.67904, p < 0.001), IL-4 (r = 0.76017, p < 0.001), and IFN-γ (r = 0.59922, p = 0.004097) in the experimental group. Moreover, the levels of nerve growth factor and hepatocyte growth factor showed a significant positive correlation (r = 0.64951, p = 0.001441) in the experimental group.Patients with idiopathic macular holes showed significant variation in aqueous humor immune response after the onset of hole formation, including the recruitment of immune cells and regulation of cytokine expression. Our findings also suggest that it is not appropriate to use patients with macular holes as the control group in studies of aqueous humor cytokine levels in ophthalmic diseases.