Laboratory diagnosis of dengue infection plays an important role for case management and disease control. Unfortunately, many of the available laboratory tests are not appropriate in terms of rapidity and simplicity. Here, we report a new format of semiquantitative dot immunoassay for detection of IgM-anti dengue antibodies in human sera employing biotinylated-dengue antigens which is simple in term of methodology and rapid in term of the test result. The results indicated that the new test is sensitive for diagnosis of secondary dengue infection. The test result can be obtained within four and a half hours.