[Background] The toxin-antitoxin system is widespread in microorganisms and plays an important role in their defense against adverse environments. [Objective] we used Yersinia pseudotuberculosis (Yptb) as the meterial to investigate the mechanism and biological functions of the Phd-Doc toxin-antitoxin system. [Methods] The Phd-Doc toxin-antitoxin system encoded in Yptb was identified by bioinformatics, and subsequently confirmed by toxicity experiment, gene expression analysis and protein interaction assay. To study the biological function of Phd-Doc toxin-antitoxin system in Yptb, the differences between the wild type strains and mutant strains were compared through antibiotic stressing, oxygen stressing and biofilm formation experiments. [Results] Bioinformatics analysis showed that a pair of Phd-Doc toxin-antitoxin system is present in Yptb, and subsequent experiments confirmed that these proteins are co-transcripted and display high affinity. The cell morphology was changed and cell growth was inhibited when the Doc toxin protein expressed in Escherichia coli, and these changes were rescued by the antitoxin protein Phd. The transcription level of phd-doc was significantly up-regulated under stress conditions, especially under the chloramphenicol stress. Deletion of phd-doc significantly affected the biofilm forming ability of Yptb but did not influence the cell growth. [Conclusion] The functional identification of the Phd-Doc toxin antitoxin system in Yptb is of great significance for a better understanding of the colonization and response mechanism of microorganisms under the changing external environment.