In rotating machines, misaligned shafts increase vibrations and friction, which can increase the energy consumption considerably and cause premature bearing and seal damage. Laser-based alignment systems can realize quick shaft alignment with high accuracy, consisting of laser transmitting unit, charge-coupled device (CCD) or position sensitive detector (PSD) receiving unit and display unit. Their resolution can reach 1 μm or higher, and the accuracy can be even up to ± (0.5%|L| + 1 resolution). In the paper, the principle of a laser-based alignment system is presented. In order to calibrate laser-based alignment systems, a set of measurement device is established. The receiving unit is fixed on a high precision one-dimensional linear stage, while the transmitting unit remains stationary. So, the relative displacement between the two units can be measured. A laser interferometer is used as the standard value of the linear stage displacement, which is traceable to the definition of meter. The layout of the calibration system is complied with Abbe's principle, reducing the measurement error. In addition, several key influencing factors for calibration are given. At last, the uncertainty of the calibration result is analyzed. The result indicates that the calibration apparatus is practical and efficient during the routine work.