Oligonucleotide microarray technology is a powerful data-mining platform and has been widely applied in biosciences. To improve the performance of assays on the oligonucleotide microarray, the factors that influence the hybridization effects such as surface chemistry, probe size, spacer length, hybridization conditions etc were intensely studied and optimized. However, it is a key problem with DNA microarrays how to generate higher fluorescent signals to improve the detection sensitivity. Two types of multiply labeled primers, termed multiply labeled linear primer and multiply labeled branched primer, were used to enhance the fluorescent signal obtained from two-dimensional DNA microarrays. The signal was intensified by increasing the number of fluorophores labeled on the target DNA segment. It was indicated that the detection limit (minimum template amount for detection) of the multiply labeled primers is about 1% of that of the singly labeled primer. Multiple labeling is an effective signal amplification method to increase the detection sensitivity of the probes in a miniaturized array format.