3-D polyacrylamide gel-based DNA microarray platforms provide a high capacity for nucleic acids immobilization and a solution-mimicking environment for hybridization. However, several technological bottlenecks still remain in these platforms, such as difficult microarray preparation and high fluorescent background, which limit their application. In this study, two new approaches have been developed to improve the convenience in microarray preparation and to reduce the background after hybridization. To control the polymerization process, solutions containing acrylamide-modified oligonucleotide, acrylamide, glycerol and ammonium persulfate are spotted onto a functionalized glass slide, and then the slide is transferred to a vacuum chamber with TEMED, so that TEMED is vaporized and diffused into the spots to induce polymerization. By applying an electric field across a hybridized microarray to remove the nonspecifically bound labeled targets, this approach can solve the problem of high fluorescent background of the gel-based microarray after hybridization. Experimental results show that our immobilization method can be used to construct high quality microarrays and exhibits good reproducibility. Moreover, the polymerization is not affected by PCR medium, so that PCR products can be used for microarray construction without being treated by commercial purification cartridges. Electrophoresis can improve the signal-to-noise significantly and has the ability to differentiate single nucleotide variation between two homozygotes and a heterozygote. Our results demonstrated that this is a reliable novel method for high-throughput mutation analysis and disease diagnosis.