Recently, we have demonstrated that DNA hybridization using acoustic streaming induced by two piezoelectric transducers provides higher DNA hybridization efficiency than the conventional method. In this work, we refine acoustic streaming system for DNA hybridization by inserting an additional piezoelectric transducer and redesigning the locations of the transducers. The Comsol® Multiphysics was used to design and simulate the velocity field generated by the piezoelectric agitation. The simulated velocity vector followed a spiral vortex flow field with an average direction outward from the center of the transducers. These vortices caused the lower signal intensity in the middle of the microarray for the two-piezoelectric disk design. On the contrary, the problem almost disappeared in the three-piezoelectric-disk system. The optimum condition for controlling the piezoelectric was obtained from the dye experiments with different activation settings for the transducers. The best setting was to activate the side disks and middle disk alternatively with 1 second activating time and 3 second non-activating time for both sets of transducers. DNA hybridization using microarrays for the malaria parasite Plasmodium falciparum from the optimized process yielded a three-fold enhancement of the signal compared to the conventional method. Moreover, a greater number of spots passed quality control in the optimized device, which could greatly improve biological interpretation of DNA hybridization data.