The design and experimental verification of a fast nucleic acid hybridization microchip using the fluidic velocity and strain rate effects was conducted. This hybridization chip was able to increase the hybridization signal 6-fold, reduce non-specific target-probe binding and background noise within 30 min, as compared to conventional hybridization methods, which may take from 4 h to overnight. Excellent correlation between experimental results and simulation analysis was obtained in this study. A detailed study of a newly designed microfluidic chip for enhancing hybridization was conducted. Three different designs of devices were fabricated and tested. Two different lengths of targets, 25-mer oligonucleotide and 1.4 kb ssDNA, were tested in this study. The hybridization efficiency can be improved by introducing velocity and extensional strain rate to the sample. This study demonstrates that the signal in the proposed method exhibits intensities 6-fold higher than those in static conditions. The necessary time for the hybridization process can be reduced from overnight to 30 min using the methods developed in this study. Experimental results also show that the strain rate provides stronger effect on hybridization than that of velocity. Combining hybridization with microfluidic concepts of velocity and strain rate effects may provide additional specificity and efficiency in nucleic acid detection and genomic study. This microfluidic hybridization chip can provide potential application in genomic study.