Technological advances in proteome research in the past few years have opened up possibilities to rapidly generate a huge amount of mass spectrometry (MS) data; Rhegmatogenous retinal detachment is the most common type of retinal detachment that is separation of the neurosensory retinal from the retinal pigment epithelium by sub-retinal fluid (SRF) defects. In this study, SRF and vitreous humor (VH) proteome have been utilised to establish a qualitative proteome analysis to investigate the origin of SRF in order to have better understanding in rhegmatogenous retinal detachment. Until now, a robust SRF proteome dataset has been lacking, which impedes precise understanding of the SRF origin. Majority group of pathways, including platelet deregulation, wound healing and complement activation, that is enriched in SRF and VH proteome are in common. A high confidence list of human plasma proteome has been investigated to study the origin of SRF, and elucidate that SRF is highly likely to be originated from VH.