Abstract: To explore genome structure characteristics, gene function and expression regulation of Euplotes amieti, high-throughput sequencing technique was used to sequence the macronucleus genome and transcriptome of Euplotes amieti. A total of 10.92 Gb data and 50287 Contigs were obtained after filtration. The GC content was 31% and the average length of exons and introns were 311.69 bp and 150 bp, respectively. The number of microchromosomes with telomeres at both ends was 27542, accounting for 54.76%, and the number of genes containing only one telomere was 6118. 38588 Unigenes were split, and the average length was 1189 bp. There were 2%—3% PRF in the total Unigenes, and most of which were +1PRF. Besides, the stop codon of Euplotes amieti also has a reconfiguration phenomenon. The stop codons are UAA and UAG, while UGA codes cysteine and selenocysteine. This was consistent with the characteristics of programmed ribosomal frameshifting and stop codon reassignments in Euplotes. 27650 genomic contigs and 38588 transcriptome Unigenes were successfully annotated. Gene function analysis showed that transcripts were significantly enriched in multiple biological processes, mainly involved cell growth and death, membrane transport, transport, and cytology. Moreover, 50 genes were randomly selected from the genome and transcriptome for PCR verification, and 95% of them were successfully verified. The results indicated that in addition to the characteristic microchromosomes and programmed frameshift, the Euplotes amieti had a large number of “combined microchromosomes” and encoded a large number of special proteins related to the perception of changes in the external environment, cell cycle, and protein expression regulation. In addition,, the gene expression may be regulated by some miRNA and zinc finger transcription factors.