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The vascular endothelial growth factor (VEGF) level in aqueous humor has been used as an indicator to monitor specific diseases in the retinal ischemic condition. For clinical diagnosis, only about 200 μL of aqueous humor can be collected from the anterior chamber before the threat of anterior chamber collapse. It is necessary to develop an inexpensive diagnostic approach with the characteristics of highly sensitive, short operation duration, and requires small clinical sample quantities. To achieve the main objective of this study, we first prepared bevacizumab to be conjugated with HRP. We then deposited 2 μL aqueous humor from patients with different diseases onto each test zone of paper-based 96-well plates. After the colorimetric results were performed via ELISA protocol, the output signals were recorded using a commercial desktop scanner for analysis. In this study, only 2 μL from the aqueous humor of each patient was required for paper-based ELISA. The mean aqueous VEGF level was 14.4 pg/mL from thirteen patients (N = 13) with senile cataract as the control. However, the mean aqueous VEGF level from other patients with proliferative diabetic retinopathy (N = 14), age-related macular degeneration (N = 17), and retinal vein occlusion (N = 10) showed VEGF increases to 740.1 pg/mL, 383 pg/mL, and 219.4 pg/mL, respectively., (Copyright © 2014 Elsevier Ltd. All rights reserved.)