An on-line measuring system, a datum calibrating method, a deviation measuring method and a computer-readable medium are provided. The datum calibrating method includes following steps. A work piece is scanned by a scanning unit to obtain a global point cloud data. A local CAD data of the work piece is obtained according to a predetermined range. A local CAD geometric feature of the local CAD data is obtained. A local point cloud data of the global point cloud data is obtained according to a local range corresponding to the predetermined range. A local scanning geometric feature of the local point cloud data is obtained. The local scanning geometric feature and the local CAD geometric feature are compared to obtain at least one spatial freedom limit. A system basis is obtained according to six spatial freedom limits, if the number of the at least one spatial freedom limit reaches six.