Here, we report TPP of hydrogel actuators. The photoinitiator 2-hydroxy-2-methylpropiophenone showed a two-photon absorption cross-section of around 4.5 GM in the excitation wavelength of 535 nm. The biocompatible material poly(ethylene glycol) diacrylate and electroactive material acrylic acid were chosen as the monomer. Reversible shape changes of these hydrogel microstructures could be achieved by the solvents and the electrical signal. Besides, their stimulus-response depended on the degree of polymerization which could be controlled by the laser power and the scanning speed. Thus, a programmable actuator with 3D geometrical configuration and tuneable stiffness was generated by TPP of hydrogels and it provided the ability to act as the valve for microfluidic channels, the operating arm for microrobots, and the smart scaffold for cell culture in vitro.