Abstract: Dust mite allergen is one of the most common allergens that cause allergies in humans. Der p 1 protein, the first allergen of house dust mite (Dermatophagoides pteronyssinus), is the most important allergen. IgE antibody specifically binding to Der p 1 can be detected in serum of over 80% allergic organism. The allergy detection kit based on Der p 1 protein can be used for allergy specific diagnosis. At present, the allergy detection kit in China is mainly depends on imports, so it is urgent to establish an efficient production system of recombinant dust mite allergen to replace imported raw materials. In this study, recombinant expression of Der p 1 was performed based on Pichia pastoris expression system. First, after codon optimization, the full-length coding gene of PreProDer p 1 was expressed in Pichia pastoris GS115, and the yield was up to 100 mg/L. Further co-expression of molecular chaperones increased the yield to 140 mg/L. And the fermentation yield of 3 L reactor was increased to 1 g/L, which was the reported highest level. Afterwards, the purification process of the recombinant protein by cation exchange chromatography and affinity chromatography was optimized, and realized the purpose protein yield reached 60.7%. Finally, the bioactivity analysis showed that the purified protein had lower antigenic activity than the commercial product. The molecular weight analysis of the protein suggested that the cleavage of PreProDer p 1 might be uneven during the secretion, and the cleavage conditions of the leader peptide requires subsequent optimization.