Multiplex PCR was used in many biological fields. Primer design and amplification conditions are critical for enhancing the efficiency of multiplex PCR. In order to improve the multiplex PCR assay, five house-keeping genes of mouse were selected to design primers for multiplex PCR analysis by using the MPprimer program, followed with optimizing the conditions for PCR reactions. Result showed that MPprimer is a valuable tool for multiplex PCR primer design. In addition, by optimizing the conditions for multiplex PCR such as the annealing temperature and extension time, the efficiency of multiplex PCR assay could be significantly improved. This work can be used to advance large-scale gene expression analysis in the post-genome era.