Vitreoretinal diseases pose significant threats to vision, often requiring vitrectomy and substitution of vitreous humor to restore ocular structure and visual function. However, existing substitutes have limitations that compromise patient outcomes. Supramolecular hydrogels, particularly peptide-based formulations, have emerged as promising alternatives due to their superior optical clarity, biocompatibility, and viscoelasticity. In this study, we designed and evaluated two peptide hydrogels, 3K-OX and 3E-OX, bearing positive and negative charges, respectively, as potential vitreous substitutes. Our in vitro findings revealed that the physicochemical properties of the negatively charged peptide hydrogel, 3E-OX, closely resembled those of the native vitreous body, exhibiting optimal light transmittance, refractive index, molecular permeability, and biocompatibility. Animal studies further confirmed the safety and biocompatibility of 3E-OX as a promising vitreous substitute. Notably, we introduced optical coherence tomography for retinal microvascular detection in non-pigmented rabbits, presenting a novel approach to evaluate the performance of intraocular tamponade materials. This work not only expands the utility of peptide hydrogels but also provides valuable insights into the design of vitreous substitutes.