In recent years, tRNA-derived small RNAs (tsRNAs) including tRNA-derived stress-induced RNAs (tiRNAs) and tRNA-derived fragments (tRFs), with specific structure and enriched in body fluids, have been found to have specific biological functions. In this paper, the biogenesis, classification, subcellular localization, and biological functions of tsRNAs were summarized. It has been proved that tsRNAs affected tumor cells in proliferation, apoptosis, migration and invasion, and played roles in regulating the occurrence and development of various tumors. In gastric cancer (GC), the imbalance of tsRNAs, such as tRF-33-P4R8YP9LON4VDP, tRF-17-WS7K092, tRF-23-Q99P9P9NDD and others, was closely related to the clinicopathological characteristics of GC patients. Some tsRNAs, such as tRF-23-Q99P9P9NDD, tRF-31-U5YKFN8DYDZDD, and tRF-27-FDXXE6XRK45 promoted the proliferation, migration and invasion of GC cells. Other tsRNAs, such as tRF-41-YDLBRY73W0K5KKOVD, tRF-18-79MP9PO4, and tRF-Glu-TTC-027 inhibited the proliferation, migration and invasion of GC cells. The tsRNAs played roles in the occurrence of GC were through several signaling pathways, such as phosphoinositide 3-kinase (PI3K)-AKT serine/threonine kinase (AKT), Wnt-beta-Catenin, and mitogen-activated protein kinase (MAPK) pathways. These findings may provide new strategies for the diagnosis and treatment of GC.