In this study, the acicular ferrite transformation behavior of a Ti–Ca deoxidized low carbon steel was studied using a high-temperature laser scanning confocal microscopy (HT-LSCM). The in situ observation of the transformation behavior on the sample surface with different cooling rates was achieved by HT-LSCM. The microstructure between the surface and interior of the HT-LSCM sample was compared. The results showed that Ti–Ca oxide particles were effective sites for acicular ferrite (AF) nucleation. The start transformation temperature at grain boundaries and intragranular particles decreased with an increase in cooling rate, but the AF nucleation rate increased and the surface microstructure was more interlocked. The sample surface microstructure obtained at 3 °C/s was dominated by ferrite side plates, while the ferrite nucleating sites transferred from grain boundaries to intragranular particles when the cooling rate was 15 °C/s. Moreover, it was interesting that the microstructure and microhardness of the sample surface and interior were different. The AF dominating microstructure, obtained in the sample interior, was much finer than the sample surface, and the microhardness of the sample surface was much lower than the sample interior. The combined factors led to a coarse size of AF on the sample surface. AF formed at a higher temperature resulted in the coarse size. The available particles for AF nucleation on the sample surface were quite limited, such that hard impingement between AF plates was much weaker than that in the sample interior. In addition, the transformation stress in austenite on the sample surface could be largely released, which contributed to a coarser AF plate size. The coarse grain size, low dislocation concentration and low carbon content led to lower hardness on the sample surface.