The comprehensive study of inclusion and microstructure characteristics in steel with TiO2 addition has been presented in this work, including wetting behavior between TiO2 and pure Fe, evaluation of inclusion agglomeration tendency, effective nuclei inclusion identification and in-situ observation of intragranular ferrite (IGF) formation. The obtained results show that TiO2 is an effective additive because it is strong wetting with liquid iron and has obvious dispersion potency. Thereafter, the effective nuclei inclusion to induce IGF formation in steel with TiO2 addition is characterized as Ti3O5 phase. The kinetic study of IGF formation using high temperature confocal laser scanning microscope shows that the fraction of IGF increases as the cooling rate increases from 3.6 to 70 °C/min and with increasing prior austenite grain size. Furthermore, it is noted that IGF morphology is refined and the onset temperatures of IGF and GBF decrease with increasing cooling rate. Finally, the onset temperature of GBF formation is higher for the steel containing a smaller grain size, however, the onset temperature of IGF formation is independent of the grain size. This work aims to give a better control of the inclusion modification as well as improving the steel quality in secondary refining and casting process, according to the concept of inclusion engineering.