Re-folded human serum albumin (rfHSA) and use thereof for anti-tumor are disclosed. The rfHSA comprises the primary amino acid sequence of naive human serum albumin, in which the rfHSA in a solution is oval shape, not fibrillar, and the naive HSA is globular. The rfHSA is used for treating cancer or a tumor in a subject in need thereof The rfHSA may also be used as a reagent for detecting the presence of a cancer cell associated with integrin β1 or serine/threonine protein kinase Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) in a tumor sample or as a reagent for inhibiting phosphorylation of Akt and ERK 1/2 in a cancer cell sample. A cell lysate of a cancer cell treated with rfHSA, a vaccine composition comprising the cancer cell lysate, and use thereof are also disclosed. Also disclosed is a method tor preparing rfHSA.