Abstract Optical microrobots are activated by a laser in a liquid medium using optical tweezers. To create visual control loops for robotic automation, this work describes a deep learning-based method for orientation estimation of optical microrobots, focusing on detecting 3-D rotational movements and localizing microrobots and trapping points (TPs). We integrated and fine-tuned You Only Look Once (YOLOv7) and Deep Simple Online Real-time Tracking (DeepSORT) algorithms, improving microrobot and TP detection accuracy by $\sim 3$ % and $\sim 11$ %, respectively, at the 0.95 Intersection over Union (IoU) threshold in our test set. Additionally, it increased mean average precision (mAP) by 3% at the 0.5:0.95 IoU threshold during training. Our results showed a 99% success rate in trapping events with no false-positive detection. We introduced a model that employs EfficientNet as a feature extractor combined with custom convolutional neural networks (CNNs) and feature fusion layers. To demonstrate its generalization ability, we evaluated the model on an independent in-house dataset comprising 4,757 image frames, where microrobots executed simultaneous rotations across all three axes. Our method provided mean rotation angle errors of $1.871^\circ$ , $2.308^\circ$ , and $2.808^\circ$ for X (yaw), Y (roll), and Z (pitch) axes, respectively. Compared to pre-trained models, our model provided the lowest error in the Y and Z axes while offering competitive results for X-axis. Finally, we demonstrated the explainability and transparency of the model’s decision-making process. Our work contributes to the field of microrobotics by providing an efficient 3-axis orientation estimation pipeline, with a clear focus on automation.