AbstractDroplet generation and transportation for biological reactions are conducted with liquid dielectrophoresis (LDEP), forming two hundred picoliter droplets and aligning them in an open environment above the micro‐machined electrodes. The generation of the dielectrophoresis signals was critically examined to actuate droplets in biological solutions without excessive Joule heating. Enzymatic reactions between β‐galactosidase and fluorescein di‐β‐D‐galactopyranoside were succeeded in manipulated droplets, which was confirmed by fluorescence imaging. These results allow us to propose the integration of LDEP actuation in high throughput biomolecular assays.