Venous leg ulcers (VLUs) are the most common type of leg ulcers with a significant socioeconomic burden due to slow healing. Cytokines may be involved in the pathogenesis of VLUs. In this systematic review, our objective was to investigate the association between cytokine levels, including growth factors, with the healing of VLUs. PubMed, Embase, Web of Science and Cochrane Library were searched from their inception to August 2021. We retrieved 28 articles investigating 38 different cytokines in 790 patients. Cytokines were most commonly investigated in wound fluid and less frequently in biopsies and serum. The studies were judged as having a moderate to high risk of bias, and the results were often inconsistent and sometimes conflicting. A meta-analysis was not performed due to clinical and methodological heterogeneities. We found weak evidence for elevated IL-1α, IL-6, IL-8, TNF-α and VEGF levels in non-healing VLUs, an elevation that declined with healing. TGF-β1 levels tended to increase with VLU healing. Other cytokines warranting further investigations include EGF, FGF-2, GM-CSF, IL-1β, IL-1Ra and PDGF-AA/PDGF-BB. We conclude that non-healing VLUs may be associated with an elevation of a palette of pro-inflammatory cytokines, possibly reflecting activated innate immunity in these wounds. There is a paucity of reliable longitudinal studies monitoring the dynamic changes in cytokine levels during wound healing.