Objective: To measure scFv antibody affinity using non-competitive enzyme-linked immunosorbent assay(ELISA). Methods:Using serial dilutions of antigen ([ Ag ], [ Ag' ] ) and antibody (anti-MAGE-Al scFv antibody), the affinity constant(Kaff) was measured by non-competitive ELISA. Two sigmoid curves of optical density (OD) versus logarithms of antibody concentrations were estimated by SPSS 10.0 software. The maximum value of OD (OD-100) of each curve was computed respectively and OD-50, half of OD-100, was obtained. Then the concentrations of antibody corresponding to OD-50 on the curves, named [Ab]t and [Ab' ]t were calculated. For [ Ag'] = 1/2 [ Ag], Kaff = 1/{2[ Ab' ]t-[ Ab]t}. Results: The affinity constant of scFv antibody was about 1.432 × 106 L/mol.Conclusion: Based on the Law of Mass Action, non-competitive ELISA method for measurement of antibody-antigen affinity constant is simple, rapid and reliable.