Abstract Although peptide‐enabled synthesis of nanostructures has garnered considerable interest for use in catalytic applications, it has so far been achieved mostly via Fmoc based solid phase peptide synthesis. Consequently, the potential of longer peptides in nanoparticle synthesis have not been explored largely due to the complexities and economic constraints of this chemical synthesis route. This study examines the potential of a 45‐amino acid long peptide expressed as fusion to green fluorescence protein (GFPuv) in Escherichia coli for use in palladium nanoparticle synthesis. Fed‐batch fermentation with E . coli harboring an arabinose‐inducible plasmid produced a product containing three copies of Pd4 peptide fused to N‐terminus of GFPuv ((Pd4) 3 ‐GFPuv). Using the intrinsic fluorescence of GFPuv, expression and enrichment of the fusion product was easily monitored. Crude lysate, desalted lysate, and an ion‐exchange enriched fraction containing (Pd4) 3 ‐GFPuv were used to test the hypothesis that high purity of the biologic material used as the nanoparticle synthesis template may not be necessary. Nanoparticles were characterized using a variety of material science techniques and used to catalyze a model Suzuki–Miyaura coupling reaction. Results demonstrated that palladium nanoparticles can be synthesized using the soluble cell extract containing (Pd4) 3 ‐GFPuv without extensive purification or cleavage steps, and as a catalyst the crude mixture is functional.