Phenylalanyl-tRNA synthetase is a key enzyme for protein synthesis in Trypanosoma. Its validation as an inhibition target will enable the development of a new generation of anti-Trypanosoma drugs. However, little is known about the isolation of the Trypanosoma Phenylalanyl-tRNA synthetase. Here we report the cloning, expression, purification and activity assay of Phenylalanyl-tRNA synthetase from Trypanosoma brucei in Escherichia coli host. We co-cloned the α-subunit and β-subunit of Phenylalanyl-tRNA synthetase from Trypanosoma brucei genomic DNA into the co-expression vector pCOLADuet. We successfully expressed the Trypanosoma brucei Phenylalanyl-tRNA synthetase in E. coli host, purified the whole enzyme by Ni-Hind affinity column and verified it by Western blotting. In addition, we tested its enzymatic activity by isotope labeling. The whole work laid a solid foundation for in vitro the screening and optimization of Trypanosoma brucei phenylalanyl-tRNA synthetase inhibitors.