When performing the inspection of subway tunnels, there is an immense amount of data to be collected and the time available for inspection is short; however, the requirement for inspection accuracy is high. In this study, a mobile laser scanning system (MLSS) was used for the inspection of subway tunnels, and the key technology of the positioning and orientation system (POS) was investigated. We utilized the inertial measurement unit (IMU) and the odometer as the core sensors of the POS. The initial attitude of the MLSS was obtained by using a static initial alignment method. Considering that there is no global navigation satellite system (GNSS) signal in a subway, the forward and backward dead reckoning (DR) algorithm was used to calculate the positions and attitudes of the MLSS from any starting point in two directions. While the MLSS passed by the control points distributed on both sides of the track, the local coordinates of the control points were transmitted to the center of the MLSS by using the ranging information of the laser scanner. Then, a four-parameter transformation method was used to correct the error of the POS and transform the 3-D state information of the MLSS from a navigation coordinate system (NCS) to a local coordinate system (LCS). This method can completely eliminate a MLSS’s dependence on GNSS signals, and the obtained positioning and attitude information can be used for point cloud data fusion to directly obtain the coordinates in the LCS. In a tunnel of the Beijing–Zhangjiakou high-speed railway, when the distance interval of the control points used for correction was 120 m, the accuracy of the 3-D coordinates of the point clouds was 8 mm, and the experiment also showed that it takes less than 4 h to complete all the inspection work for a 5–6 km long tunnel. Further, the results from the inspection work of Wuhan subway lines showed that when the distance intervals of the control points used for correction were 60 m, 120 m, 240 m, and 480 m, the accuracies of the 3-D coordinates of the point clouds in the local coordinate system were 4 mm, 6 mm, 7 mm, and 8 mm, respectively.