To quantitatively describe the effect of out-of-focused-plane displacements on the in-focused-plane micro-vision motion-tracking accuracy， a method based on an optical microscope， an industrial camera， and a spatial nano-positioning stage is proposed in this work. First， the formation mechanism of out-of-focused-plane displacements was analyzed. The variance function was employed to evaluate the sharpness of the image and search for the best-focused plane. The searched plane was used as the baseline to calculate out-of-focused-plane displacements. Second， a spatial nano-positioning stage was designed with multiple degrees of freedom. The end-effector served as the target to be tracked by the micro-vision. When generating in-focused-plane displacements， controllable and measurable out-of-focused-plane displacements were also produced simultaneously and synchronously by the end-effector. Third， the gray-value-based template matching approach was selected and combined with the region-of-interest method. This approach served as a representative micro-vision motion-tracking algorithm. Finally， the prototype of the nano-positioning stage was fabricated， and the micro-vision motion-tracking experimental system was constructed. Capacitive sensors were employed as evaluation tools. The degradation values of the micro-vision motion-tracking accuracy when excited by different out-of-focused-plane displacements were quantified. The experimental results indicate that out-of-focused-plane displacements directly contribute to accuracy degradation. The displacements and degradation were significantly correlated. The area of the proposed viewing field was 76.1 μm×63.7 μm， the sampling frequency was 15 Hz， and the gray-value-based template-matching approach was employed. When the out-of-focused-plane displacement reached （7.7±2.5） μm or larger， the micro-vision system was rendered inoperable.