Jominy end-quench test samples from CF53 were used for studying the relationship of microstructural changes with the magnetic Barkhausen noise (BN) response. As the Barkhausen noise method is sensitive to both stress and microstructural state, it can be applied for material characterization. This study presents observations from BN measurements with different sensors and from different locations (as-quenched and ground) on the sample surface. Detailed microstructural characterization with a scanning electron microscope and a transmission electron microscope was carried out to correlate the BN responses with the microstructural features. In addition, residual stresses were measured by X-ray diffraction. The results indicate that the ground surface displayed mainly the effect of the grinding compressive stress state, while the as-quenched surface had variations due to higher microstructure sensitivity. An important finding of the results was that the sensitivity of BN to different surface conditions varied: The BN response in the ground area was mainly generated by both the residual stress and the microstructural effect, whereas for the as-quenched surface the microstructural effect was more evident.