A poly(dimethylsiloxane) (PDMS) patterning method based on a photoresist lift-off technique to make an electrical insulation layer with selective openings is presented. The method enables creating PDMS patterns with small features and various thicknesses without any limitation on the designs and without the need for complicated processes or expensive equipment. Patterned PDMS layers were created by spin-coating liquid phase PDMS on top of a substrate having sacrificial photoresist patterns, followed by a photoresist lift-off process. The thickness of the patterned PDMS layers could be accurately controlled (6.5–24 µm) by adjusting processing parameters such as PDMS spin-coating speeds, PDMS dilution ratios and sacrificial photoresist thicknesses. PDMS features as small as 15 µm were successfully patterned and the effects of each processing parameter on the final patterns were investigated. Electrical resistance tests between adjacent electrodes with and without the insulation layer showed that the patterned PDMS layer functions properly as an electrical insulation layer. Biocompatibility of the patterned PDMS layer was confirmed by culturing primary neuron cells on top of the layer for up to two weeks. An extensive neuronal network was successfully formed, showing that this PDMS patterning method can be applied to various biosensing microdevices. The utility of this fabrication method was further demonstrated by successfully creating a patterned electrical insulation layer on flexible substrates containing multi-electrode arrays.