Background: Evidence shows that the microbiome plays an important role in allergic diseases. The associations between the conjunctival microbiome dysbiosis and ocular allergy remain unknown. This study investigates the characteristics of the conjunctival microbiome in allergic conjunctivitis (AC). Methods: We performed metagenomic shotgun sequencing on conjunctival samples from 48 healthy subjects and 39 patients with AC. The AC group comprised 21 patients with seasonal allergic conjunctivitis or perennial allergic conjunctivitis (SAC/PAC) and 18 patients with vernal keratoconjunctivitis (VKC). Results: We identified a signature microbiome associated with the dysbiosis of conjunctival microbiota on the ocular surface of patients with AC. Interestingly, SAC/PAC and VKC harbor distinct microbial communities. Specifically, Brevibacterium aurantiacum is enriched in SAC/PAC, whereas a group of Streptococcus species and Haemophilus parainfluenzae are enriched in VKC. Conclusion: Dysbiosis of conjunctival microbiome is found on the ocular surface of AC patients. These results suggest a new microbial mechanism of AC and provide a potential target for AC treatment.