RNA secondary structure elements in the mRNA 3'-untranslated regions (3'UTR) play important roles in post-transcriptional regulation. RNA structure elements in the viral RNA provide valuable model for studying diverse regulation mechanisms. Herpesvirus genomes are double-stranded DNA with GC-rich sequences, which can be transcribed into abundant GC-rich RNAs. It is valuable to explore the structures and function of those GC-rich RNAs. We identified a G2-quadruplex-forming sequence named PQS18-1 in the 3'UTR of the unique immediate early gene of Pseudorabies virus (PRV), an important member of Alphaherpesvirinae subfamily. The RNA PQS18-1 was folded into parallel G-quadruplex structure, enhancing gene expression. Both non-G-quadruplex mutant and G3-quadruplex mutant in the 3'UTR showed lower gene expression level than the wildtype G2-quadruplex. TMPyP4 destroyed PQS18-1 G2-quadruplex and suppressed gene expression, accordingly reducing PRV replication by one titre in the PK15 cells at 24 h post infection. Our findings indicated that the RNA G2-quadruplex in 3'UTR was essential for high expression of IE180 gene, and it could be a specific post-transcription regulation element in response to small molecules or other macromolecules. This study discovers a novel RNA G2-quadruplex in the 3'UTR of an immediate early gene of alphaherpesvirus and provides a new nucleic acid target for anti-virus drug design.