In this research, we propose an Error Separation Technique (EST) based on optical fiber sensors for on-machine straightness error measurement. Twofiber optic Fabry-Perot inter-ferometers have been developed serving as two displacement sensors. The displacement dis-tance is computed according to the reflected spectrum from interferometers, which can achieve a sub-micrometer resolution. The two-point method has been employed to separate the straightness error of the slides and the profile error of afine-polished standard block . The spacing distance between two interferometers is determined by the diameter of optical fibers so that the EST's resolution has the potential to reach the sub-millimeter scale. In the experiment, the straightness error has been measured on a commercially available com-puter numerical control machine tool, and the measurement has been conducted on its x-axis. The spacing distance between two opticalfiber sensors is 1.5 mm which equals the EST's resolution along the machine tool's x-axis. The separated profile error of the mea-sured standard block is around 30 mu m which has been verified by a high precision Coordi-nate Measurement Machine (CMM). The magnit u d e of the separated straightness error is around 40 mu m. This technique is flexible and simple to be conducted, which can contribute to the micro-machine tool calibration and other straightness error applications.