Acoustic droplet ejection (ADE) is a noncontact technique for micro-liquid handling (usually nanoliters or picoliters) that is not restricted by nozzles and enables high-throughput liquid dispensing without sacrificing precision. It is widely regarded as the most advanced solution for liquid handling in large-scale drug screening. Stable coalescence of the acoustically excited droplets on the target substrate is a fundamental requirement during the application of the ADE system. However, it is challenging to investigate the collision behavior of nanoliter droplets flying upward during the ADE. In particular, the dependence of the droplet's collision behavior on substrate wettability and droplet velocity has yet to be thoroughly analyzed. In this paper, the kinetic processes of binary droplet collisions were investigated experimentally for different wettability substrate surfaces. Four states occur as the droplet collision velocity increases: coalescence after minor deformation, complete rebound, coalescence during rebound, and direct coalescence. For the hydrophilic substrate, there are wider ranges of Weber number (We) and Reynolds number (Re) in the complete rebound state. And with the decrease of the substrate wettability, the critical Weber and Reynolds numbers for the coalescence during rebound and the direct coalescence decrease. It is further revealed that the hydrophilic substrate is susceptible to droplet rebound because the sessile droplet has a larger radius of curvature and the viscous energy dissipation is greater. Besides, the prediction model of the maximum spreading diameter was established by modifying the droplet morphology in the complete rebound state. It is found that, under the same Weber and Reynolds numbers, droplet collisions on the hydrophilic substrate achieve a smaller maximum spreading coefficient and greater viscous energy dissipation, so the hydrophilic substrate is prone to droplet bounce.