The microwell plate/microtiter plate is among the most widely used tools in immune assays. In this paper, we report on a sensitive method for enhancing fluorescence emission detection by simply adding several droplets of an immiscible organic compound into the microwells before detection. To prove the concept, human IgA was determined on a microwell plate using this droplet enhanced fluorescence (DEF) detection method. An obvious enhancement in fluorescence was observed. The detection limit (LOD) was about 1/20 times and the sensitivity was 4 times greater than that without droplets. To prove the use of the method for disease diagnosis, the IgG of measles in human plasma was determined using the proposed DEF method. A LOD of around 1/5 times and a sensitivity of 4 times the DEF were easily achieved compared to ELISA with a conventional fluorescence detection.