A novel femtosecond laser patterning technique is reported to construct a three-dimensional (3D) wettability pattern on a superhydrophobic TiO2 nanotube array (TNA) surface in one-step. A 3D binary TNA pattern with extremely high contrast for microfluidic manipulators and biomedical scaffolds is used to guide droplet transportation and human mesenchymal stem cell site-selective growth, respectively.