Micro hot embossing process is a cost-effective method for parallel replication of polymer microstructures. To study the effect of hot embossing and demoulding parameters on the replication accuracy of the polymer microstructure, densely patterned and high-depth channels on 50mm×50mm polymer substrates were fabricated by hot embossing in this paper. Experimental results showed that the replication accuracy of the microstructure increased with the increasing of hot embossing temperature and the thickness of polymer substrates. It can be found that demoulding became more difficult when replication accuracy increased. The reason was that higher replication accuracy resulted in larger contact area between the replica and the embossing mold. The demoulding problem could be solved by rising demoulding temperature. However, overhigh demouding temperature (110°C) would lead to round corners at the edges of the ribs. Experimental results also showed that very small channel widths and depths errors (less than 1.2%) of the microstructures with 2mm thickness substrates could be achieved, when embossing and demoulding temperatures were set to 120°C and 100°C, respectively.