In France and Finland, farmer’s lung disease (FLD), a hypersensitivity pneumonitis common in agricultural areas, is caused mainly by Eurotium species. The presence of antibodies in patients’ serum is an important criterion for diagnosis. Our study aimed to improve serological diagnosis of FLD by using common fungal particles that pollute the farm environment as antigens. Fungal particles of the Eurotium species were observed in handled hay. A strain of Eurotium amstelodami was grown in vitro using selected culture media, and antigen extracts from sexual (ascospores), asexual (conidia) and vegetative (hyphae) forms were made. Antigens were tested by Enzyme Linked Immunosorbent Assay (ELISA), testing for immunoglobulin G antibodies from the sera of 17 FLD patients, 40 healthy exposed farmers and 20 non-exposed controls. The antigens were compared by Receiver Operating Characteristic analysis and a threshold was then established. Ascospores contained in asci enclosed within cleistothecia were present in 38% of the hay blades observed; conidial heads of aspergilli conidial heads of aspergilli were less present. The same protocol was followed to make the three antigen extracts. A comparison of results for FLD patients and exposed controls showed the area under the curve to be 0.850 for ascospore antigen, 0.731 for conidia and 0.690 for hyphae. The cut-off we determined, with the standard deviation for measures taken into account, showed 67% for sensitivity and 92% for specificity with the ascospore antigen. In conclusion, serological diagnosis of FLD by ELISA was improved by the adjunction of ascospore antigen.