Venous leg ulcers (VLUs) are common and cause significant morbidity and poor quality of life. There is a poor understanding of the biology underlying non-healing VLUs. VLU exudates may reflect the underlying wound microenvironment. This systematic review aims to identify potentially diagnostic and/or prognostic protein biomarkers within VLU fluid/exudates reported in the literature. A systematic review was reported according to PRISMA guidelines. MEDLINE and Embase databases were searched up to 31st March 2024. Full text, primary studies in English reporting on proteins identified in VLU fluid/exudate were included. Two independent reviewers performed the abstract and full-text screen. Additional publications were identified by searching the references of included studies. 46 studies were identified, with nine comparing healing and non-healing VLUs. Cytokines (e.g., IL-1a, IL-1ra, IL-6, eotaxin, GM-CSF, PDGF, VEGF) and proteins involved in extracellular matrix (ECM) homeostasis (e.g., MMP-7, MMP-10, MMP-13, TIMP-4) were significantly increased in non-healing compared to healing VLUs. Collagen subunits (PICP and PIIINP) significantly increased as the VLU healed. Inflammatory proteins (e.g., complement type 6, S100A8, S100A9) and ECM proteins (e.g., fibronectin, lumican) were found to be increased in non-healing VLUs compared to acute surgical wounds. Altered levels of specific proteins in wound exudates may be indicative of healing and non-healing VLUs. Further work is essential to elucidate a comprehensive protein phenotype that may help early identification and prognostication of non-healing VLUs.