The present invention relates to the method of measuring a biological ligand (1) in a sample containing an amylase (1) derived from a higher animal. This method is characterized by employing a combination of an amylase (2) not contained in the sample and an antibody against the ligand (1) or a ligand (2) capable of reacting with the antibody and an amylase inhibitor of which inhibitory activity against the amylase (1) is stronger than that against the amylase (2). The method of the invention utilizes the emphasis of steric hindrance of the amylase (2) reaction which is carried out at the boundary between solid phase and liquid phase and the specific removal of the influence of the amylase (1) by using the above amylase inhibitor. This method is suitable as a clinical test for the determination of physiological substances and trace components in a humoral fluid such as serum.