The development of an accurate and efficient method for detecting missing bolts in engineering structures is crucial. To this end, a missing bolt detection method that leveraged machine vision and deep learning was developed. First, a comprehensive dataset of bolt images captured under natural conditions was constructed, which improved the generality and recognition accuracy of the trained bolt target detection model. Second, three deep learning network models, namely, YOLOv4, YOLOv5s, and YOLOXs, were compared, and YOLOv5s was selected as the bolt target detection model. With YOLOv5s as the target recognition model, the bolt head and bolt nut had average precisions of 0.93 and 0.903, respectively. Third, a missing bolt detection method based on perspective transformation and IoU was presented and validated under laboratory conditions. Finally, the proposed method was applied to an actual footbridge structure to test its feasibility and effectiveness in real engineering scenarios. The experimental results showed that the proposed method could accurately identify bolt targets with a confidence level of over 80% and detect missing bolts under different image distances, perspective angles, light intensities, and image resolutions. Moreover, the experimental results on a footbridge demonstrated that the proposed method could reliably detect the missing bolt even at a shooting distance of 1 m. The proposed method provided a low-cost, efficient, and automated technical solution for the safety management of bolted connection components in engineering structures.