Platanus acerifolia, London plane tree, a significant source of airborne allergens, is widely grown in Shanghai and other cities in China. In recent decades, little has been known regarding the influence of the allergens on sensitizing the population in the Shanghai area. The aims of this study were to purify and characterize the two major allergens and to confirm the immunological activities of these pollen allergens in Shanghai. Crude extract was purified with a HiTrap SP column and a Sephedex G75 column. Immunodetection was performed with ELISA and immunoblotting. Following gel proteolytic digestion and mass spectrometry, the tandem MS (MS/MS) peptide mass fingerprint was obtained and the MASCOT search engine was used to identify the peptide. The accession number of the interesting homologous data and all the sequence information was acquired by an internet database and the evolutionary trees were drawn with Mega 4.0 software. Two proteins with molecular weights of 43 kDa and 18 kDa were purified from P. acerifolia pollen extract. The purified proteins were identified as pollen allergen Pla a 1 and Pla a 2 via mass spectrometry. The proteins have immunological activities with human IgE antibodies. According to the ELISA results, 12% (5/41) of the subjects were sensitive to Pla a 1 and 9% (4/41) were sensitive to Pla a 2. Pla a 1 and Pla a 2 are thus important allergens for patients with an allergic reaction to P. acerifolia pollen in Shanghai.