Factor IX is a vitamin K-dependent coagulation factor which undergoes several post-translational modifications for its proper function. We established the Chinese hamster ovary cells expressing the recombinant human coagulation factor IX (rhFIX). In order to maximize the expression of rhFIX in serum-free suspension culture, we evaluated the effect of several metal ion concentrations including Ca2+ and Mg2+ in the cell serum-free culture medium. A high production FIX CHO cell line was screened and the highest clotting activity was obtained in 2 mM Ca2+, combined with 1 mM Mg2+ showed an excellent improvement compared with culture without any metal ion addition, the peak FIX activity was 1.36 IU/ml compared with 0.62 IU/ml. It means a higher level of fermentation process and the CHO cell line is a promising alternative for recombinant FIX manufacturing.