An object to be solved by the present invention is to determine the dissociation constant of an analyte molecule immobilized on a metal surface and a molecule that interacts therewith in surface plasmon resonance (SPR) analysis via an analytical method that produces a low noise level (i.e., a noise width of a reference chip), small baseline fluctuations (i.e., signal changes of a reference chip), and highly reliable results of measurement. The present invention provides a method for determining a dissociation constant of an analyte molecule immobilized on a metal surface and a molecule that interacts therewith, wherein changes in surface plasmon resonance signals are measured by using a surface plasmon resonance measurement device comprising a flow channel system having a cell formed on a metal film and a light-detecting means for detecting the state of surface plasmon resonance by measuring the intensity of a light beam totally reflected on the metal film; wherein a change in surface plasmon resonance signals is measured in a state where the flow of the liquid has been stopped, after the liquid contained in the above flow channel system has been exchanged; and wherein the dissociation constant is determined based on the results of measurement of signal changes.