Background: Pseudorabies virus (PRV) is a highly infectious pathogen that affects a wide range of mammals and imposes a significant economic burden on the global pig industry. The viral envelope of PRV contains several glycoproteins, including glycoprotein E (gE) and glycoprotein B (gB), which play critical roles in immune recognition, vaccine development, and diagnostic procedures. Mutations in these glycoproteins may enhance virulence, highlighting the need for updated vaccines. Method: This review examines the functions of PRV gE and gB in vaccine development and diagnostics, focusing on their roles in viral replication, immune system interaction, and pathogenicity. Additionally, we explore recent findings on the importance of gE deletion in attenuated vaccines and the potential of gB to induce immunity. Results: Glycoprotein E (gE) is crucial for the virus’s axonal transport and nerve invasion, facilitating transmission to the central nervous system. Deletion of gE is a successful strategy in vaccine development, enhancing the immune response. Glycoprotein B (gB) plays a central role in viral replication and membrane fusion, aiding viral spread. Mutations in these glycoproteins may increase PRV virulence, complicating vaccine efficacy. Conclusion: With PRV glycoproteins being essential to both vaccine development and diagnostic approaches, future research should focus on enhancing these components to address emerging PRV variants. Updated vaccines and diagnostic tools are critical for combating new, more virulent strains of PRV.