Since the discovery of the first miRNA in 1993, numerous studies have focused on their biogenesis, their functions on regulating a diversity of cellular processes, and the molecular mechanisms underlying their regulatory activity. The critical roles they play during pathogenesis have also been explored. With the advancement on next-generation sequencing, new classes of small RNA with distinct functions have been discovered. Among them, tRNA derived fragment (tsRNAs) have become a center of studies due to their similarity to miRNA. In this review, we summarized the biogenesis of miRNA and tsRNAs, the molecular mechanisms of their functions, and their important roles during the development of diseases. The similarity and difference between miRNA and tsRNAs were also discussed.