In this paper, we propose a simple method for analyzing interface interactions in bead-based bioassay. Although bead-based bioassay enables simple and rapid quantitative evaluation of target substances, non-specific adsorption between the beads or between the beads and a substrate affects the reliability of measurement. Conventional methods for evaluating non-specific adsorption include a method in which a magnetic force or shearing force is applied to beads to prevent non-specific adsorption, but the experimental system is complicated. Therefore, a simpler evaluation method would be useful for carrying out a systematic study to find optimal conditions for the suppression of non-specific adsorption. Therefore, we devised a new adhesion behavior analysis method based on simple force application by gravity and the observation of Brownian motion. The analysis results obtained by the devised method were considered on the basis of DLVO (Derjaguin–Landau–Verwey–Overbeek) theory, which is the adhesion theory of solid surfaces, and the main factors contributing to non-specific adsorption were identified. Furthermore, changes in adhesion behavior with ionic strength, bead size, and reaction time were investigated. The results provide useful information for optimizing the experimental system in bead-based bioassay and are expected to improve the reliability of bioassay.