Thaumetopoea pityocampa causes allergies and skin and ocular lesions. No commercial tools are currently available for the clinical diagnosis of this allergy. We aimed to develop an in vitro method for the diagnosis of this allergy to avoid patients undergoing in vivo tests with insect extracts. Recombinant Tha p 2 was produced and used in an ELISA validated with 15 allergic patients. Subsequently, 42 subjects recruited from a random sampling cross-sectional study were analysed. The ELISA sensitivity and specificity were 93.3% and 100%, respectively, for the allergic patients and 71.4% and 95.3%, respectively, for the epidemiological study. The positive ELISA results correlated with the skin prick test areas with the whole body and the setae extracts. Professional exposure and short latency of symptoms onset were risk factors for a positive result in the ELISA. In conclusion, our ELISA is very useful for T. pityocampa allergy diagnosis and for epidemiologic testing.