Purpose To explore the relationships between the aqueous and vitreous levels of vascular endothelial growth factor-A (VEGF-A), interleukin-8 (IL-8), placental growth factor (PlGF) and erythropoietin (EPO) in proliferative diabetic retinopathy (PDR) and neovascular glaucoma (NVG). Methods Aqueous and vitreous samples were obtained from patients with PDR and NVG during surgery. Aqueous and vitreous concentrations of VEGF-A, IL-8, PlGF and EPO were measured via enzyme-linked immunosorbent assay. Results No correlation between the aqueous and vitreous levels of VEGF-A, IL-8, PlGF or EPO was found in both the PDR and the NVG eyes. Aqueous VEGF-A was significantly higher in the NVG group (317.55 +/- 36.25 pg/ml, n = 15) than that in the PDR group (256.23 +/- 46.11 pg/ml, n = 17, P < 0.001). The level of VEGF-A in aqueous (317.55 +/- 36.25 pg/ml, n = 15) was significantly higher than that in vitreous (224.74 +/- 60.32 pg/ml, n = 15, P < 0.001) in NVG patients. The level of IL-8 in aqueous (76.55 +/- 10.88 pg/ml, n = 17) was significantly higher than that in vitreous (63.55 +/- 10.74 pg/ml, n = 17, P = 0.001) in PDR patients. The level of EPO in aqueous (18.62 +/- 2.87 mIU/ml, n = 15) was significantly higher than that in vitreous (15.97 +/- 3.11 mIU/ml, n = 15, P = 0.022) in NVG patients. The ratio of aqueous versus vitreous for VEGF-A was significantly higher in the NVG group (1.475 +/- 0.289, n = 15) than that in the PDR group (0.996 +/- 0.227, n = 17, P < 0.001). Conclusion Aqueous levels of VEGF-A, IL-8, PlGF and EPO do not correlate with vitreous levels of those proteins. The relationship between protein levels in aqueous humor and vitreous might be dependent on different disease status or protein types investigated.