Long hairpin RNA (lhRNA) construct-induced gene silencing facilitates the study of gene function in plants and animals, but constructing multiple lhRNA vectors using traditional approaches is both time-consuming and costly. Also, most of the existing approaches are based on sequence-specific cloning of individual sequences, and are therefore not suitable for preparing hpRNA libraries from a pool of mixed target sequences. Here we describe a rolling-circle amplification (RCA)-mediated hpRNA (RMHR) construction system suitable for generating libraries of lhRNA constructs from any gene of interest or pool of genes. Using RMHR we successfully generated a lhRNA library from a Arabidopsis cDNA population containing known and unknown genes, with an average size of 500–800 bp for the inverted-repeat inserts. To validate the RMHR system, lhRNA constructs targeting the β-glucuronidase (GUS) gene were tested using Agrobacterium infiltration and shown to be effective at inducing GUS silencing in tobacco leaves. Our results indicate that the RMHR technique permits rapid, efficient and low-cost preparation of genome-wide lhRNA expression libraries.