Serial sectioning by focused ion beam milling for three-dimensional electron backscatter diffraction (3D-EBSD) can create surface damage and amorphization in certain materials and consequently reduce the EBSD signal quality. Poor EBSD signal causes longer data acquisition time due to signal averaging and/or poor 3D-EBSD data quality. In this work a low kV focused ion beam was successfully implemented to automatically polish surfaces during 3D-EBSD of La- and Nb-doped strontium titanate of volume 12.6 × 12.6 × 3.0 μm. The key to achieving this technique is the combination of a defocused low kV high current ion beam and line scan milling. The line scan was used to restrict polishing to the sample surface and the ion beam was defocused to ensure the beam contacted the complete sample surface. In this study 1 min polishing time per slice increases total acquisition time by approximately 3.3% of normal 3D-EBSD mapping compared to a significant increase of indexing percentage and pattern quality. The polishing performance in this investigation is discussed, and two potential methods for further improvement are presented.