In this study, we analyzed the transcriptional activity and interacting proteins of AtWRKY61 transcription factor through dualluciferase(LUC), yeast twohybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments. qRTPCR was used to analyze the response characteristics of AtWRKY61 to various abiotic stress. They lay the foundation for further revealing the function and molecular regulation mechanism of AtWRKY61. Subcellular localization through green fluorescence protein (GFP) shows that AtWRKY61 is localized in the nucleus only. A dualLUC assay in Arabidopsis protoplasts and yeast experiment reveal that AtWRKY61 has transcriptional repression activity. qRTPCR results indicate that transcription of AtWRKY61 responded to multiple abiotic stresses and hormone stimuli, suggesting AtWRKY61 may participate in multiple signaling pathways. Y2H test demonstrated that AtWRKY61 interacted with itself and two closely related paralogs AtWRKY9 and AtWRKY72, which were further confirmed by BiFC. This implies that AtWRKY61 might perform its transcriptional repression function via forming a complex with itself and two other closely related homologs.