To investigate the evolution law of inclusions in 42CrMo-S steel, this paper samples and analyzes the steel during its refining process as well as the head and tail billets. An oxygen and nitrogen analyzer, a scanning electron microscope (SEM) equipped with energy-dispersive X-ray spectrometry (EDS), and an ASPEX automatic inclusion scanning electron microscope are employed to analyze the cleanliness level of the molten steel in the refining stage and the head and tail billets. The results demonstrate that the total oxygen content at the end of LF slagging is 10.2 ppm, indicating that the refining slag has an excellent deoxygenation effect. During the RH refining process, the total oxygen content of the molten steel diminishes to less than 10 ppm and reaches 6.3 ppm at end-RH. The nitrogen content in the molten steel gradually increases during the smelting process and attains 65 ppm at end-RH. Upon arrival at LF, pure Al2O3 plays the role of the primary inclusions in the molten steel. Afterwards, the pure Al2O3 inclusions transform into Mg-Al spinel-type inclusions, Al2O3-MgO-CaO inclusions, and Al2O3-CaO inclusions. The number of CaS-type inclusions in the steel reaches the maximum after feeding the S wire. In the RH refining stage, the percentage of inclusions with a size less than 5 μm is maintained above 90%. Finally, the cleanliness level of the head and tail billets (the start and end of a casting sequence) is analyzed, and it is recommended that the cut scrap length for the head billet is 0.3 m and the reasonable cutting scrap length for the tail billet is 1 m.