Abstract: The diffusion characters of particles constrained between two shortly separated flat walls are different from those of particles in the infinite space. In this paper, constrained diffusion of an isolated micro-sphere (about 1μm in diameter, Polystyrene) confined between two parallel flat walls was studied using video microscopy combined with optical tweezers by measuring the distance of Brownian motion of a sphere within a period of time. We also developed a method of measuring the distance of vertical motion of a sphere based on the correlation analysis of the sphere image, which decreases the measurement error, by which the relation between diffusion character of a sphere and its distance to the bottom of the sample cell was experimentally studied.