Biological libraries are powerful tools for discovery of new ligands as well as for identification of cellular interaction partners. Since the first development of the first biological libraries in form of phage displays, numerous biological libraries have been developed. For the development of new ligands, the usage of synthetic oligonucleotides is the method of choice. Generation of random oligonucleotides has been refined and various strategies for random oligonucleotide design were developed. We trace the progress and design of new strategies for the generation of random oligonucleotides, and include a look at arising diversity biases. On the other hand, genomic libraries are widely employed for investigation of cellular protein-protein interactions and targeted search of proteomic binding partners. Expression of random peptides and proteins in a linear form or integrated in a scaffold can be facilitated both in vitro and in vivo. A typical in vitro system, ribosome display, provides the largest available library size. In vivo methods comprise smaller libraries, the size of which depends on their transformation efficiency. Libraries in different hosts such as phage, bacteria, yeast, insect cells, mammalian cells exhibit higher biosynthetic capabilities. The latest library systems are compared and their strengths and limitations are reviewed.