The tRNA-derived RNA fragments (tRFs) and tRNA halves (tiRNAs) are derived from mature transfer RNAs (tRNAs) or precursor tRNAs through specific cleavage at different sites. They are belonged to small non-coding RNA molecules widely existing in prokaryotic and eukaryotic transcriptome. tRFs are classified into tRF-5s, tRF-3s and tRF-1s. tRF-5s and tRF-3s are generated from the cleavage between D-ring and anticodon loop to 5′-end, and T-ring to 3′-end of mature tRNAs, respectively. tRF-1s are from the 3′ trailer fragment of precursor tRNAs. The sizes of tRFs are 14-30 nt. tiRNAs, 29-50 nt in length, are divided into 5′ tiRNAs and 3′ tiRNAs. They are generated within the anti-codon loop. tRFs and tiRNAS have a variety of biological functions. They can be not only stress-activated signal molecules but also coordinators of gene expression. Moreover, tRFs and tiRNAs are associated with the occurrence of a variety of human diseases such as cancers, neurodegenerative diseases, hereditary metabolic diseases, and infectious diseases. Thus, they may become a new type of biomarkers for the diagnosis of these diseases. In this paper, the classification of tRFs and tiRNAs, their biological functions, and their relationships with human diseases were reviewed.