Diagnosis of canine aspergillosis is difficult using currently available methods. It often passes unnoticed or is diagnosed in the later phases of the disease. We developed an ELISA technique to detect anti-Aspergillus antibodies in canine serum using an Aspergillus antigenic mycelial extract, which could then be used for the diagnosis of canine aspergillosis. We used a cut-off of X + 3SD obtained from 20 control sera. The test was performed on 46 dogs with lesions indicating possible aspergillosis and gave nine positive results: one systemic mycosis, two discospondylitis, one uveitis, two bronchopulmonary processes and three rhinitis. We compared this methodology with the PLATELIA technique in the follow-up of the affected dogs, obtaining the same limitations as in the diagnosis of human aspergillosis. We consider our ELISA technique using sera samples a speedy, safe and reliable method which enables us to follow up the evolution of the disease and the efficacy of the therapy chosen. A definitive diagnosis must still take into account the results of other tests such as clinical examination, radiographic studies, endoscopy and biopsy.