Muscle-invasive bladder cancer (MIBC) leads to a large societal burden. Recently, tRNA-derived small RNAs (tsRNAs), a novel type of noncoding RNA (ncRNAs), have been identified. However, the expression patterns and functions of tsRNAs in MIBC have not yet been identified. Here, RNA sequencing, bioinformatics, and quantitative reverse transcription- polymerase chain reaction (qRT-PCR) were used to screen the expression profiles and predict the potential roles of tsRNAs in MIBC. Of 406 tsRNAs differentially expressed in MIBC tissues, 91 tsRNAs were significantly differentially expressed. Then, four candidate tsRNAs, tiRNA-1:34-Val-CAC-2, tiRNA-1:33-Gly-GCC-1, tRF-1:32-Gly-GCC-1, and tRF-+1:T20-Ser-TGA-1, were selected. Next, a bioinformatics analysis showed the potential target genes and tsRNA-mRNA network. The most significant and meaningful terms of gene ontology were the positive regulation of the phosphate metabolic process, lamellipodium, and protein-cysteine S-acyltransferase activity in the biological process, cellular component, and molecular function, respectively. In addition, the top four pathways were predicted by the Kyoto Encyclopedia of Genes and Genomes database (KEGG). Finally, qRT-PCR demonstrated a similar expression pattern compared to sequencing data for the candidate tsRNAs. In short, we find differential expression profiles and predict that tiRNA-1:33-Gly-GCC-1, tRF-1:32-Gly-GCC-1, and tRF-+1:T20-Ser-TGA-1 are very likely to engage in the pathophysiological process of MIBC via regulating the target genes in the key pathways.