Abstract We present a novel process for the lift-off transfer of a pyrolyzed polymer onto a low thermal tolerance substrate. Pyrolyzed polymer is a promising carbon material for applications of electrochemical bio-sensors. However, it is difficult to form pyrolyzed polymer on low thermal tolerance substrates because annealing temperatures greater than 600°C are necessary to transform dielectric polymers to conductive carbon materials. From a practical viewpoint, it is essential to form metal patterns such as Au electrodes with the transferred pyrolyzed polymer. Therefore, we propose a process for the transfer of the Au/pyrolyzed polymer combined layer. The materials and the thickness of the barrier layer, parameters of the post-annealing process, and surface activation are investigated to ensure successful transfer using the proposed process. Because it became possible to transfer pyrolyzed polymer even onto heat workable polymer substrates such as cyclic olefin polymer films, the developed technology can be employed in lab-on-a-chip applications such as biosensors on polymer based microfluidic chips.