This invention discloses a method for real-time 3D tracking and localization of microscale targets in a bright-field microscopic environment, relating to the fields of image recognition and reconstruction technology. The method includes inputting continuous microscopic images into a target detection model, outputting the target's 2D position, regressing the defocus height and direction from a
single frame of local images using a fine-grained ROI-based defocus regression model, directly using the model output to drive a 2D motion platform and a focusing device, fusing the 2D position information of each frame with the corresponding defocus axis information, and incorporating the position information of the previous frame as a priori using a
temporal correlation mechanism to achieve continuous correlation of the microscale target in the time dimension, thereby stably reconstructing the target's 3D position and outputting a smooth real-time motion trajectory. This invention achieves real-time 3D localization and stable tracking of microscale targets under single-view bright-field microscopic conditions, ensuring both positioning accuracy and
system real-time performance and accuracy.