In this paper, an on-machine error calibration method, covering error modeling and measurement, is proposed to evaluate and compensate the errors caused by the mechanical and optical system equipped in the micromachining center using the femtosecond laser. Through preliminary tests by dicing silicon wafer, it has revealed that the squareness, laser beam misalign and focal position offset, are the main causes to result in the inaccuracy of micromachining. Consequently, an error modeling method is proposed to evaluate the error distribution in the workspace, and hereafter a comprehensive error vector of the laser beam, combining the squareness errors of Z-axis with the laser beam misalign, is generated by the variable substitution method. Subsequently, an increment error model in the instant local coordinates is established to satisfy the requirement of the programming method commonly used in the laser machine tools. Furthermore, a series of holes and grooves are machined on the femtosecond laser micromachining center to validate the proposed approach and model. The machining dimensions including diameters, distances and angles, are measured on-machine to identify the squareness errors, laser beam misalign and focal position offset according to the proposed error model. Finally, the experimental results show that, comparing to the uncompensated tests, the machining accuracy has been significantly improved with the proposed method.