Affinity reagents of high affinity and specificity are very useful for studying the subcellular locations and quantities of individual proteins. To generate high-quality affinity reagents for human Lyn tyrosine kinase, a phage display library of fibronectin type III (FN3) monobodies was affinity selected with a recombinant form of the Lyn SH3 domain. While a highly specific monobody, TA8, was initially isolated, we chose to improve its affinity through directed evolution. A secondary library of 1.2 × 109 variants was constructed and screened by affinity selection, yielding three variants, two of which have affinities of ~ 40 nM, a 130-fold increase over the original TA8 monobody. One of the variants, 2H7, displayed high specificity to the Lyn SH3 domain, as shown by ELISA and probing arrays of 150 SH3 domains. Furthermore, the 2H7 monobody was able to pull down endogenous Lyn from a lysate of Burkitt's lymphoma cells, thereby demonstrating its utility as an affinity reagent for detecting Lyn in a complex biological mixture.