Ribosome display is a powerful engineering research tool for the high-throughput selection of peptides or proteins, which results in the generation of high-performance binders against nearly any antigen of interest. As a cell-free display system, ribosome display has been well developed with many outstanding achievements for over 20 years. Compared with other related display techniques, ribosome display shows unique advantages and development prospects. This tool has been successfully exploited for the selection of functional and specific binders in vitro. Here, we describe methods for the construction and characterization of ribosome-displayed natural nanoantibody libraries, by which specific nanoantibody screening can be performed.