The Epstein-Barr nuclear antigen 1 (EBNA1) is a crucial protein expressed by the Epstein-Barr virus (EBV). The EBNA1 is necessary for the replication and transcriptional regulation of latent gene expression of the EBV. Therefore, it is connected with some diseases, especially malignancies. Previous studies have shown that chalcone potentially inhibited the EBV virus; therefore, in this study a series of chalcones were screened in silico toward EBNA1 by the use molecular docking and molecular dynamics simulation. The results suggested that chalcone 3a displayed significantly greater binding affinity than the reported anti-EBV agents. The EBNA1 residues K477, I481, N519, K586, and T590 contributed mainly for the chalcone 3a binding at the recognition helix site. Altogether, this chalcone might serve as a lead compound acting against EBNA1.