In order to study the effect of Ca and sulfur contents on the characteristics of inclusions, industrial experiments using 95CrMo steel were conducted. SEM-EDS detections and stereological analysis were used to probe the characteristics of inclusions, including their compositions, morphologies, size, number density, and distribution. The results indicate that there were mainly three types of inclusions in 95CrMo steel billets with 6–18 ppm Ca and 30–100 ppm S: inclusions with single-phased morphology mainly composed of oxides; isolated MnS/CaS-only inclusions; inclusions with multi-phased morphology. The three-dimensional inclusion size distribution suggests that there were more Type-1 inclusions with a small size in low S containing steels. The average diameter of all types of inclusions increased with increasing Ca or S content in 95CrMo steel, indicating that the formation of MnS and CaS coarsened their size. The density distribution of inclusions indicates that the more inclusions there are, the more easily they aggregate and collide. Moreover, it is presumably concluded that the formation of sulfide in the outer layer of oxide inclusions weaken the attraction between oxide inclusions. The equilibrated transformation and formation of inclusions during the cooling process of 95CrMo steel was discussed based on thermodynamic calculation. The equilibrated transformation trajectory of inclusions in 95CrMo steel during the cooling process was Ca2SiO4 + MgO → Ca3MgSi2O8 → Spinel + CaS, which was corresponding to the detected results. The precipitation regular of sulfide was obtained. The formation mechanism for three types of inclusions was discussed.