Dental pulp stem cells (DPSCs) are a unique population of odontogenic undifferentiated mesenchymal stem cells with strong self-renewal ability and multi-lineage differentiation potential existing in the dental pulp tissue. They could differentiate into osteoblasts, chondrocytes, odontoblasts, adipocytes, nerve cells, muscle cells, and hepatocytes under specific stimulation conditions, providing a novel clinical idea for their application in the treatment of various tissue defects and repairs. However, in the process of passage and expansion in vitro, DPSCs inevitably exhibit decreased stemness, such as slowed proliferation rate, cell senescence, and pluripotency decline, which seriously impede their application in tissue engineering. How to overcome these deficiencies and maintain the stemness of DPSCs have received extensive attention in tissue engineering research. In this article, the research progress on the significance, methods (including low-temperature preservation, culture dimensions, hypoxic environment, application of cytokines, etc.), and molecular mechanisms of stemness maintenance of DPSCs was reviewed.