Anti-VEGF therapy is commonly used to treat proliferative diabetic retinopathy (PDR), but the exact mechanism of VEGF signaling is not fully understood. Using data-independent acquisition mass spectrometry (DIA-MS), we analyzed proteomic changes in aqueous humor (AH) samples collected before and one week after intravitreal ranibizumab (IVR) treatment from 10 PDR patients to discover potential biomarkers. Resultantly, 875 proteins were quantified and 26 proteins were significantly altered in response to IVR treatment in PDR. Further investigation through gene ontology (GO) and pathway analysis revealed that these differentially expressed proteins were primarily involved in extracellular matrix (ECM) and platelet degranulation signaling. Protein-protein interaction analysis highlighted five hub proteins (COL3A1, DPT, VEGFA, SPP1, SERPING1) that were found to be ECM components. Enzyme-linked immunosorbent assay (ELISA) confirmed the decreased levels of VEGFA and increased levels of DPT proteins after IVR treatment in another 8 samples of AH in 4 PDR patients. Our study provided novel insights into aqueous proteins of PDR following IVR treatment. Targeting the ECM pathway, particularly the elevation of DPT protein, may provide a deeper understanding of the anti-VEGF resistance and VEGF signaling in PDR.