Replication of micro structures on a polymer substrate employing two-stage hot embossing is described in this paper. Linear channels with rectangular cross section were fabricated on a master mold of a brass plate by means of a computer numerical control (CNC) machining center using a micro end mill. In the first embossing process, the master mold with concavity features was embossed on a polycarbonate (PC) plate in order to make a “polymer replication mold”. Polytetrafluoroethylene (PTFE) was chosen as a material of polymer substrate used in the second embossing process. The replicated structure on PTFE substrate was observed and each dimension of its cross-sectional shape was also measured in order to assess the replication accuracy. According to the results, it was found that the depth of a replicated channel was near 90 % of that on the master mold. But roundness at each corner could be also found due to the insufficiency of plastic flow. Furthermore the deformation of the structures on a replication mold of PC before and after 2nd hot embossing process was also examined. As a result, it was found that the deformation ratio at temperatures below 145 oC was roughly smaller than 10 %.