Fibrosis is a pathological process in which parenchymal cells are necrotic and excess extracellular matrix (ECM) is accumulated due to dysregulation of tissue injury repair. Thymosin β4 (Tβ4) is a 43 amino acid multifunctional polypeptide that is involved in wound healing. Prolyl oligopeptidase (POP) is the main enzyme that hydrolyzes Tβ4 to produce its derivative N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) which is found to play a role in the regulation of fibrosis. Accumulating evidence suggests that the Tβ4-POP-Ac-SDKP axis widely exists in various tissues and organs including the liver, kidney, heart, and lung, and participates in the process of fibrogenesis. Herein, we aim to elucidate the role of Tβ4-POP-Ac-SDKP axis in hepatic fibrosis, renal fibrosis, cardiac fibrosis, and pulmonary fibrosis, as well as the underlying mechanisms. Based on this, we attempted to provide novel therapeutic strategies for the regulation of tissue damage repair and anti-fibrosis therapy. The Tβ4-POP-Ac-SDKP axis exerts protective effects against organ fibrosis. It is promising that appropriate dosing regimens that rely on this axis could serve as a new therapeutic strategy for alleviating organ fibrosis in the early and late stages.