Traditional off-line measuring systems find it difficult to measure micro-structured workpieces which have a large volume and heavy weight, such as micro-structured patterned precision roller drums. This paper proposes an autostereoscopy-based three-dimensional (3D) measuring method and develops an innovative measuring system for the 3D on-machine measurement of the micro-structured surfaces, an Autostereoscopy-based Three-Dimensional On-machine Measuring (ATDOM) system. The ATDOM system is compact and capable of fast data acquisition and high accuracy in 3D computational reconstruction of complex surfaces under different measuring environments. A prototype ATDOM system is experimentally verified through a series of measurement experiments conducted on a precision machine tool. The results indicate that the ATDOM system provides an important means for efficient and reliable on-machine measurement of micro-structured surfaces.