Abstract: Pesudocohnilembus persalinus is a harmful parasite found in marine fish. Using SMART technology, a full-length cDNA library of P. persalinus was constructed. Titer of the primary library was 3.3×10⁶ cfu·mL^-1, and the library capacity was 1.5×10⁷ cfu with a recombination rate of 97%. A PCR amplification on 24 random clones revealed that the inserted cDNA fragments ranged from 300-2 000 bp, with an average length over 500 bp. Subsequently, 1 200 clones were selected from the library for EST sequencing. As a result, 1 032 high-quality ESTs were obtained and assembled into 292 unigenes including 102 contigs and 190 unique ESTs with a redundancy rate of 71.7%. In a blast analysis against NCBI database, 215 of the unigenes were homologous to the corresponding genes of ciliates with free-living and parasitic lifestyle, such as Tetrahymena thermophila, Paramecium tetraurelia, Ichthyophthirius multifiliis, Cryptocaryon irritans, Oxytricha trifallax, etc. But, only 134 ESTs had been annotated. The remainders were hypothetical protein genes. The results obtained from this study would substantially facilitate the molecular cloning and characterization of the functional genes in P. persalinus.