We report a method for the preparation of multi-domain liquid crystal (LC) alignment layers on polyimide surfaces with laser-induced periodic surface structures. Atomic force microscopy confirmed that the laser-induced microstructures could be easily erased and re-constructed by repeated laser exposure. The microstructures demonstrated excellent abilities to align LCs in a multi-domain way. The intricate micropatterning of LCs with high resolution was obtained. The opto-electric properties of the LC cells were also determined. In addition, the method is available to a wide range of polymeric substrates, including flexible ones such as poly(ethylene terephthalate).