MGL_1304 secreted by Malassezia globosa is contained in human sweat and induces histamine release from basophils in patients with atopic dermatitis (AD) at a high positive rate. The aims of this study were to establish the enzyme-linked immunosorbent assay (ELISA) measuring specific immunoglobulins against MGL_1304 and to investigate the levels of these immunoglobulins in sera of patients with various allergic diseases. Purified MGL_1304 from human sweat (QRX) and recombinant MGL_1304 (rMGL_1304) were prepared for ELISA. To quantify the amount of MGL_1304-specific immunoglobulins, the standard serum was created by pooling sera of 20 patients with AD whose basophils released histamine in response to QRX. A monoclonal antibody which exhibited the highest neutralizing ability against QRX was established as Smith-2, and used as a capture antibody for the assay of QRX-specific IgE. A total of 156 subjects normal controls (n = 23), AD (n = 63), cholinergic urticaria (CU) (n = 24), bronchial asthma (n = 32), and allergic rhinitis (n = 14) were enrolled in this study. ELISA methods to quantify the specific IgE, IgG and IgG4 against MGL_1304 in sera were successfully established. Levels of QRX-specific IgE in sera of patients with AD and CU were significantly higher than those of normal controls. Moreover, the levels of QRX-specific IgE and rMGL_1304-specific IgE in patients with AD were significantly correlated with their disease severities. These ELISA methods to quantify the specific immunoglobulins against MGL_1304 are easy and useful means to assess allergy to MGL_1304. MGL_1304 contained in sweat is an important antigen for patients with AD and CU.