The honey bee is the most essential pollinator and a key contributor to the natural ecosystem. There are numerous ways for thousands of bees in a hive to communicate with one another. Individual trajectories and social interactions are thus complex behavioral features that can provide valuable information for an ecological study. To study honey bee behavior, the key challenges that have resulted from unreliable studies include complexity (high density of similar objects, small objects, and occlusion), the variety of background scenes, the dynamism of individual bee movements, and the similarity between the bee body and the background in the beehive. This study investigated the tracking of individual bees in a beehive environment using a deep learning approach and a Kalman filter. Detection of multiple bees and individual object segmentation were performed using Mask R-CNN with a ResNet-101 backbone network. Subsequently, the Kalman filter was employed for tracking multiple bees by tracking the body of each bee across a sequence of image frames. Three metrics were used to assess the proposed framework: mean average precision (mAP) for multiple-object detection and segmentation tasks, CLEAR MOT for multiple object tracking tasks, and MOTS for multiple object tracking and segmentation tasks. For CLEAR MOT and MOTS metrics, accuracy (MOTA and MOTSA) and precision (MOTP and MOTSP) are considered. By employing videos from a custom-designed observation beehive, recorded at a frame rate of 30 frames per second (fps) and utilizing a continuous frame rate of 10 fps as input data, our system displayed impressive performance. It yielded satisfactory outcomes for tasks involving segmentation and tracking of multiple instances of bee behavior. For the multiple-object segmentation task based on Mask R-CNN, we achieved a 0.85 mAP. For the multiple-object-tracking task with the Kalman filter, we achieved 77.48% MOTA, 79.79% MOTSP, and 79.56% recall. For the overall system for multiple-object tracking and segmentation tasks, we achieved 77.00% MOTSA, 75.60% MOTSP, and 80.30% recall.