Small RNAs and enzymes that provide their biogenesis and functioning are involved in the organism development and coordination of biological processes, including metabolism, maintaining genome integrity, immune and stress responses. In this review, we focused on the role of small RNA biogenesis proteins in determining the aging and longevity of animals and human. A number of studies have revealed that changes in expression profiles of key enzymes, in particular proteins of the Drosha, Dicer and Argonaute families, are associated with the aging process, as well as with some age-related diseases and progeroid syndromes. Down-regulation of small RNA biogenesis proteins leads to global alterations in the expression of regulatory RNAs, disruption of key molecular, cellular and systemic processes, which leads to a lifespan shortening. In contrast, overexpression of Dicer prolongs lifespan and improves cellular defense. Additionally, the role of small RNA biogenesis proteins in the pathogenesis of age-related diseases, including cancer, inflammaging, neurodegeneration, cardiovascular, metabolic and immune disorders, has been conclusively evidenced. Recent advances in biomedicine allow using these proteins as diagnostic and prognostic biomarkers and therapeutic targets.