cDNA microarray is widely used to identify and quantify different gene-expression for large-scale analysis. In order to extract microarray data precisely from microarray images, robust image processing is indispensable. Most microarray image processing methods are still semiautomatic because of the variations between different arrayers and spots properties. This study attempts to propose an automatic spot detection algorithm using mathematical morphology methods. Watershed transform is the main technique for our spot automatic detection method. To avoid the over-segment problem brought on the sensitive to noises of gradient image used in watershed algorithm, we preprocessed the image using morphological opening operator. Internal and external marker for allocating watershed immersion positions were used to reduce the number of image segments. The result showed that our morphological methods achieved automatic spot detection on non-supervised environment.