Optical motion capture systems are devices used to track and capture target movements and obtain the position and attitude of the target in real time. In practical tasks such as UAV obstacle avoidance, vehicle-machine co-positioning and multi-machine co-operation, optical motion capture systems are required to provide reliable and high-precision positioning coordinates for targets. On the one hand, the optical motion capture system provides high-precision positioning coordinates for the target to assist in tasks such as navigation and obstacle avoidance; on the other hand, the optical motion capture system provides high-precision positioning true values for the target to verify the accuracy of the positioning and navigation algorithms. However, the actual positioning accuracy of the optical motion capture system is often much lower than the manufacturer's nominal accuracy due to factors such as the camera distribution, number of cameras in the optical motion capture system and vibrations in the field. Therefore, it is urgent and necessary to verify the actual positioning accuracy of optical motion capture systems in large scenes. Especially in large scenes, optical motion capture systems suffer from scattered camera distribution, a small number of cameras in the common viewing area, and long camera-target distances, resulting in inaccurate positioning. If the actual positioning accuracy of the optical motion capture system is significantly different from the nominal accuracy, the performance of the motion capture system will be greatly reduced and it will be difficult for the motion capture system to perform its actual function. In general, the positioning accuracy of the optical motion capture system is mainly tested in small spaces, and the positioning accuracy of the system is often assessed in terms of repeated trajectory accuracy, resulting in the actual positioning accuracy of optical motion capture systems in real scenes, especially in large scenes, being unknown or inaccurate. To solve the problem of precision detection of the optical motion capture system in large scenes, a method of calibrating the positioning accuracy of the optical motion capture system in large scenes using total station is proposed. First, select several common points evenly in the four sub-regions where the optical motion capture system is located. Second, the concentric target ball workpiece of the same size is used to solve the problem that the measuring points of the total station and the optical motion capture system are inconsistent. Third, the real value and measured value of the common point are obtained through the measurement of the total station and optical motion capture system and the position and posture transformation matrix between the total station coordinate system and the optical motion capture system coordinate system is calculated based on robust least squares iterative algorithm for Rodrigues matrix. Finally, the positioning accuracy of the optical motion capture system in the sub-region and the whole region is calculated under the unified coordinate system. Meanwhile, the external parameter calibration of the measurement system and the Vicon system is realized based on the above method. The experimental results show that the positioning accuracy of the four sub-region optical motion capture systems is 2.385 mm, 0.877 mm, 1.787 mm, 2.890 mm respectively, and the positioning accuracy of the optical motion capture system in the whole region is 8.126 mm. It shows that the positioning accuracy of the optical motion capture system is significantly reduced in large scenes, and the feasibility and effectiveness of using the total station to calibrate the optical motion capture system and external parameters of the motion capture system are verified. With the increasing application demand and positioning accuracy requirements for optical motion capture systems, the proposed method is an inspiration and reference for future calibration of optical motion capture systems in large and complex scenes.