The invention relates to a trypanosoma brucei phenylalanyl-tRNA synthetase preparation method, belonging to the technical field of biology and the preparation method comprises the following specific steps: separately designing primers, amplifying the alpha subunit and beta subunit genes of trypanosoma brucei phenylalanyl-tRNA synthetase; cloning the alpha subunit and beta subunit genes into prokaryotic expression vector pET21a(+), building pET21a(+)-PheRS alpha and pET21a(+)-PheRS beta expression vector; subcloning the operon of alpha subunit into pET21a(+)-PheRS beta, forming recombinant co-expression vector pET21a(+)-PheRS alpha beta; transforming the recombinant co-expression vector pET21a(+)-PheRS alpha beta into colibacillus BL21(DE3)RIPL, inducting expression with IPTG; collecting the supernate of the lysed bacteria solution, purifying proteins and obtaining the trypanosoma brucei phenylalanyl-tRNA synthetase. The invention builds the recombinant plasmid of the trypanosoma brucei phenylalanyl-tRNA synthetase (PheRS), and expresses and purifies trypanosoma brucei phenylalanyl-tRNA synthetase protein, thus laying a foundation for the screening of antiparasitic agent.