The present invention belongs to the field of genetic engineering drugs, and provides a mutein MuR5S4TR of TRAIL, and a preparation method and use thereof. The N-terminal positions 2-10 of the amino acid sequence of the mutein consist of the transmembrane peptide sequence RRRRR (R5) and the binding sequence AVPI of the apoptosis inhibitor XIAP, the positions 11-169 are the TRAIL protein peptide segment (123-281 aa), and the specific sequence is as shown in SEQ ID NO: 2.