New diseases in marine animals are emerging at an increasing rate, yet methodological limitations hinder characterization of viral infections. Viral metagenomics is an effective method for identifying novel viruses in diseased animals; however, determining virus pathogenesis remains a challenge. A novel anellovirus (Zalophus californianus anellovirus, ZcAV) was recently reported in the lungs of captive California sea lions involved in a mortality event. ZcAV was not detected by PCR in the blood of these animals, creating the inability to assess the prevalence of ZcAV in live sea lions. This study developed an enzyme-linked immunosorbent assay (ELISA) to detect antibodies to ZcAV in sea lion serum. To assess ZcAV prevalence, paired serum and lung samples (n = 96) from wild sea lions that stranded along the California coast were tested through ELISA and PCR, respectively. Over 50% of the samples tested positive for ZcAV by ELISA (34%), PCR (29%), or both (11%) assays. ZcAV is prevalent in stranded wild sea lion populations and results suggest that PCR assays alone may grossly underestimate ZcAV exposure. This ELISA provides a tool for testing live sea lions for ZcAV exposure and is valuable for subsequent studies evaluating the potential pathogenicity of this anellovirus.