An improved and simplified protocol for DNA immobilization was developed to enhance DNA-DNA hybridization on microwell plates. Target DNA was immobilized by simple dry-adsorption. Efficiencies of DNA immobilization and retention were enhanced 1.4-6.5 times and 4.2-19.6 times, respectively, compared with a conventional method. The overall hybridization efficiency was increased 3.1-5.2 times. This simple new protocol can reduce the consumption of scarce DNA samples.