Abstract: Total RNA and subsequent mRNA were isolated from E tenella sporozoites A library of Oligo(dT)-primed cDNA with added directional EcoR I /Hind Ⅲ linkers was constructed and ligated to the EcoR I /Hind Ⅲ arms of the screen vector, the recombinant phage DNA was packaged by using PhageMaker packaging extracts, resulting in a primary cDNA library. The library was proved to be a good quality by the control tests. The cloning efficiency was evaluated and the length of the cDNA fragment was assayed by PCR. Using the amplified library as template DNA, a pair of primers were designed according to the sequence of the E. tenella 3-1E, then the gene was amplified by PCR. The results showed that the cDNA expression library of E. tenella sporozoites was constructed and the 3-1E gene was amplified successfully. The capacity of the cDNA library was 4×106 pfu/mL and the length of inserts was about 100-3 000 bp. It is helpful in the further study on screening related genes.