The major transient expression platforms for recombinant therapeutic proteins (RTPs) are Chinese hamster ovary (CHO) cells. Most improving strategies have been focused on expression promotion at the transcriptional level in CHO cells. How to promote RTPs yields through translation regulation remains unclear. This study investigated the effects of the 5’ untranslated region (UTR) characteristics on the recombinant protein expression of CHO cells. And used Ribo-seq to characterize regions on the 5’UTR that can promote translation initiation efficiency by complementary pairing with ribosomal RNA. Some elements located in 5’UTR and its characteristics differentially affected the translation of main ORF, resulting in a 1.5-fold increase in protein expression in CHO cells. This study presents a 5’UTR sequence analysis method and provides a new idea to break the bottleneck of RTPs yield from the rarely considered translation enhancement.