In this research, the effect of different types of commercial tundish refractory working layers (gunning mix, prefabricated board (PB), and dry mix) on the formation of blowhole defect in continuous casting steel billets was investigated. To protect the tundish body, three refractory layers are applied on its metallic shell including a safety layer, a permanent layer, and a working layer. Due to the direct contact of molten metal and slag with the tundish working layer, this layer plays an important role in guaranteeing the quality of steel. In the current research, the amount of molten metal hydrogen pick-up due to contact with the abovementioned tundish working layers was measured by a Hydris device. The hydrogen content of all the melts increased after transferring from ladle to tundish, but the rising rate strongly depended on the type of tundish working layer with PB having a minimum amount of hydrogen pick-up. To characterise the working layers, their chemical and mineralogical composition as well as their thermal behaviour up to 1400 °C were evaluated using X-ray fluorescence, X-ray diffraction, and thermogravimetry-differential scanning calorimetry techniques. The results showed that olivine is a main constituent of all the working layers. Also, the amount and quality of olivine (especially the amount of its serpentinization) have a great effect on the formation of blowhole defects in the steel billets.