R-loops are formed during transcription when the nascent RNA generated by RNA polymerases hybridizes with its complementary DNA template, giving rise to a region of DNA∶RNA hybrid and a displaced single-stranded DNA. R-loops are stable structures that have important beneficial physiological functions, but also could pose a threat to genomic stability. Unscheduled R-loops induce cell cycle checkpoint activation, DNA damage, and chromosome rearrangement in mammalian cells. R-loops expose unstable single-stranded DNA, which is prone to transcription-related mutations and recombination. R-loops may also directly block DNA replication, leading to DNA double strand breaks. Abnormal accumulations of R-loops have been found in some syndromes, human neurological disorders, and cancers. On the other hand, R-loops also play positive roles in physiological processes, such as epigenetic modification, DNA repair, gene regulation and mitochondrial stability. R-loops forming on transcription-termination regions, promote RNA polymerase pausing before termination. R-loops are regulated delicately in cells. Collisions between replication and transcription cause accumulation of R-loops. Replication stress, DNA damage and RNA Pol II pausing also induce R-loop formation. To resolve R-loops when they form, cell evolve numerous dissolution mechanisms. Ribonuclease RNase H1 and RNase H2 bind to R-loops and then catalyze the cleavage of RNA. Helicases, such as SETX, DHX9, DDX21, unwind the RNA from the R-loops. Defects in RNA processing factors, chromatin modulators, DNA repair proteins, cause accumulation of R-loops, suggesting they are involved in R-loop regulations. To detect R-loops, several methods have been developed and are mainly based on the S9.6 antibody and the HBD domain of RNase H1, however, both of them possess some issues. Understanding the regulatory mechanisms of R-loop formation and clearance could help us better know how cells maintain genomic stability and prevent disease development. In this review article, we summarized functions and regulations of R-loops. We also discussed methodologies used to detect R-loops. Finally, we proposed some future perspectives of R-loop research.