Olive pollen is the main cause of pollinosis in Mediterranean countries. The immunological analysis of Ole e 1, the major allergen of Olea europaea, has usually carried out by means of ELISA (Enzyme-Linked ImmunoSorbent Assay). However, most published works only specify the methodology related to antigen quantifications, but not the related to protein extraction. Furthermore, the results obtained are not compared with different buffers or modifications of them. The main aim of this study is to obtain an optimized and reproducible ELISA protocol for quantifications of Ole e 1 in the atmosphere. The study of Ole e 1 allergen and olive pollen in the atmosphere of Malaga (Spain) was carried out by means of an automatic multi-vial cyclonic sampler and a Hirst volumetric pollen trap, respectively. ELISA was tuned up on the basis of previously published protocols to quantify this allergen. Variations in the concentrations of capture and detection antibodies, as well as in the buffers used to carry out the extraction, were evaluated. The highest protein extraction was obtained when a modified buffer was applied. The correlation analysis between daily pollen concentrations and allergen quantifications showed highly significant values. The ELISA protocol, together with the buffer combination proposed in this work, considerably reduced the concentrations of capture and detection antibodies used for quantifying Ole e 1 in the atmosphere, allowing detect this allergen even in days in which the airborne pollen concentration was very low or null.