Background: Locusts as model systems are widely used in many biological laboratories worldwide. Occupational exposure to locusts induces a high prevalence of allergic sensitization. However, knowledge on occupational locust allergens remains unclear. This study aimed to identify a novel allergen from locusts that causes occupational allergies. Methods: We conducted a 20-year retrospective survey of 94 persons using questionnaires and a cross-sectional survey of 57 persons using questionnaires and immunological tests for occupational allergies in long-term locust laboratories. We identified the major allergens by immunoblotting and analysed them by LC-MS/MS. The allergenicity of the major allergen proteins was assessed by specific IgE (sIgE) detection, immunoblotting and ELISA inhibition assays. Results: The retrospective survey indicated that the frequency of occupational allergies was relatively low (13.8%), while the cross-sectional survey showed a higher frequency (40.4%). The symptoms in most allergic males were allergic rhinitis and asthma, while females showed higher prevalence of atopic dermatitis. Occupational exposure for 2-3 h per day or continuing one and half years obviously increased the allergy risk. We identified the hexamerin-2 protein as a major allergen in locusts. Purified hexamerin-2 protein achieved approximately 60% serum IgE reactivity with locust protein extract. The potential for cross-reactivity with cockroaches was indicated by sequence alignment of the hexamerin-2 protein and allergens of cockroaches. Conclusion: Occupational exposure is an important risk factor for locust allergy. The hexamerin-2 protein of locusts as a major allergen in occupational allergy was identified for the first time.