Low temperatures are the most significant abiotic stressor for the conservation and production of Phalaenopsis in non-tropical areas. CBF/DREB1 transcription factors play an important role in the plant abiotic stress response. In this study, 31 DREB family members were identified in the Phalaenopsis genome. Expression pattern analysis showed that the expression of different PaDREB members varied among tissue sites. PaDREB1D was isolated from Phalaenopsis aphrodite, and multiple sequence alignment showed that PaDREB1D belonged to the A1 subgroup of the DREB family and was localized in the nucleus. PaDREB1D overexpression in protocorm-like bodies of Phalaenopsis reduced cell damage during low-temperature stress, increased antioxidant enzyme activity, and enhanced the low-temperature tolerance of protocorm-like bodies. The results of this study provide a theoretical basis for breeding for cold resistance and investigating the molecular mechanisms related to low-temperature responses in Phalaenopsis.