Two sequence-inversed probes were microarrayed on glass slides to study the hybridization efficiency with their DNA targets. A fluorescence laser scanner and an atomic force microscope (AFM) were utilized to investigate the efficiency in different hybridization cases and their corresponding depth changes on the chips. The sequences of two targets were designed to be fully complementary to their shared DNA probe in a coaxial stacking configuration. In other words, after the first DNA target is hybridized (pre-hybridizing) onto the probe, the second one is stacked onto the non-hybridized region of the same probe. The pre-hybridizing and the second DNA targets were distinguished by two distinct fluorescent dyes. The enhancement of the hybridization efficiency was investigated through the comparison between the stacking and individual hybridization configurations. AFM was used to measure the depths of two probes at different steps of hybridization. The results indicated that the depths increased as the hybridization proceeded. Probe#1, pre-hybridizing close to the chip surface, obtained a thicker depth than the other probe pre-hybridizing away from the chip surface, Probe#2. A hypothesis was proposed to explain how the depth variation was associated with the observed hybridization efficiency.