MicroRNAs (miRNAs) are short, noncoding RNAs that are key players in regulating genes after they are transcribed, by targeting mRNAs for degradation or inhibiting translation. The biogenesis of miRNAs is a process which occurs in multiple steps, first involving transcription into the genome, followed by processing by the Drosha and Dicer enzymes along the way, and finally by incorporation into the RNA-induced silencing complex (RISC) in order to carry out their regulatory functions. miRNAs are important for a variety of biological activities, including cell differentiation, proliferation, apoptosis, and immune response. Dysregulation of miRNA expression and function is linked to multiple diseases, particularly cancer, neurodegenerative diseases, and metabolic syndromes. Advances in sequencing technologies and bioinformatics have led to a better understanding of miRNA biogenesis and function, as well as their potential as both biomarkers and therapeutic targets. However, some challenges remain, for example, the development of efficient delivery systems, off-target effects, and the complexities of miRNA-mRNA interactions. This chapter will highlight important concepts regarding miRNA biogenesis and its implications for health and disease, as well as future opportunities for miRNA-based diagnostics and therapeutics.