Based on transcriptome re-sequence, the translationally controlled tumor protein gene from Aphelenchoides besseyi, named Ab–TCTP, was cloned using RT–PCR and RACE. The coding sequence was 810 bp with 546 bp open reading frame (ORF), and encoded a protein with 181 amino acids. The putative protein molecular weight was 20 930 and its theoretical isoelectric point (PI) was 4.68, belonged to a stable proteins, and located in nucleus without signal peptide and transmembrane structure. The amino acid sequence of the Ab–TCTP protein in function areas tended to be conservative, contained TCTP conservative domain structure, and belonged to the TCTP superfamily. The results from multiple sequence alignment showed that the query coverage between protein Ab–TCTP and Caenorhabditis elegans TCTP (XP_001891979.1) was 100%, with a similarity of 78%. The results from phylogenetic tree analysis indicated that the Ab–TCTP protein was very close to TCTP of C. elegans. The results from in situ hybridization test indicated that the enhanced hybridization signals were mainly observed in dorsal esophageal gland of A. besseyi. The pharmaceutical experiment with rotenone showed that the value of LC50 was 2.20 μg/mL after 24 h, and the results from real-time quantitative PCR presented that the expression of Ab–TCTP was up-regulated, which the expression quantity in 24 h was larger than that in 12 h. Therefore, the Ab–TCTP gene was speculated to participate in the stress response of A. besseyi.