We demonstrate that the efficiency of DNA hybridization could be improved by introducing elevated temperature in the hot region and higher velocities in the cold region of a microfluidic chip. Compared with the conventional methods, this hybridization microchip was shown to increase the hybridization signal 4.6-fold within 30 minutes using a 1.4 kb target DNA as the test material. The increase in fluorescence intensity was apparent when the temperature was higher than 82°C, and the fluorescence intensity reached an asymptotic value as T > 90°C. A mathematical model was proposed to relate the fluorescence intensity of DNA hybridization with the temperature of hot region and the velocity of cold region. Based on these results, the new hybridization chip with the processes of temperature and velocity differences will provide additional efficiency in DNA detection.