Next-generation deep sequencing (NGS) technology represents a powerful and innovative approach to profile small RNA. Currently, there are a number of large-scale and benchtop sequencing platforms available on the market. Although each platform is relatively straightforward to operate, constructing cDNA libraries can be the most difficult part of the NGS workflow. Constructing quality libraries is essential to obtaining a successful sequencing run of high-quality reads and coverage. The quality and yield of RNA affect hybridization and ligation of sequencing adapters. In the field of biomarker discovery, there has been an interest in profiling exosomal RNA from biological fluids. However, very little RNA yield is obtained when extracting RNA from exosomes, thus making library construction difficult. Here, this protocol describes an optimized protocol for constructing small RNA libraries from low yields of RNA, in particular, extracted from exosomes isolated from biological fluids.