Avian aspergillosis, most often caused by Aspergillus fumigatus, is a common and devastating disease affecting a range of bird species. Early diagnosis is difficult and often unreliable. The current study evaluated the utility of measuring (1→3)-β-D-glucan (BG) concentrations in avian plasma samples to aid in the diagnosis of aspergillosis. We evaluated a commercially available BG assay (Fungitell®, Beacon Diagnostics) using 178 plasma samples from naturally infected, experimentally infected, and aspergillosis-free birds. Although there was variation in BG concentration, as reflected by high standard deviations, seabirds with confirmed aspergillosis had the highest mean BG concentrations (M  =  3098.7 pg/dl, SD  =  5022.6, n  =  22) followed by companion avian species and raptors with confirmed aspergillosis (M  = 1033.8 pg/dl, SD  =  1531.6, n  =  19) and experimentally infected Japanese quail (Coturnix japonica; M  =  1066.5 pg/dl, SD  =  1348.2, n  =  17). Variation in severity of disease, differences among species of birds with and without disease, and also different levels in environmental exposure likely contribute to the differences among avian groups. The overall sensitivity and specificity of the BG test for diagnosis of aspergillosis in birds was 60.0 and 92.7%, respectively, with an overall optimized avian cut-off value of ≥461 pg/dl for positive disease. Our findings suggest that, although BG concentrations are highly variable between and within different avian groups, it could serve as a useful adjunctive diagnostic test for aspergillosis that is applicable to multiple avian species in some settings, particularly as a negative predictor of infection.