We present a compensation method for the sensor gain difference when a mixed sequential two-probe method (MTPM) is applied for precision measurements of straightness error. According to theoretical analysis of the effect of sensor gain error, much of the measurement error comes from the sensor gain difference. Also, measurement error caused by misalignment of the straight-edge is dramatically amplified by the sensor gain difference. Therefore, the measurement error in MTPM can be significantly reduced by compensating for the sensor gain difference. To compensate for sensor gain difference, we propose a very simple approach that uses sensor outputs for two different alignment conditions of the straight-edge. To verify the proposed compensation method, experiments were performed with a hydrostatic linear motion table. Experimental results show that the gain difference of the two sensors used in the experiments was about 3.4%. Before compensation, the deviation in the calculated straightness error depending on the alignment conditions was 1.30 μm, which was reduced to 0.08 μm after compensation. The compensated measurement results were compared with results obtained by a reversal method. The maximum deviation between the two methods was 0.22 μm. These results demonstrate that the proposed compensation method is very effective for the measurement of straightness error with sub-micron accuracy and in addition it is practical because compensation can be performed on the actual measurement setup.