Pycnoporus sanguineus and its intracellular protein extracts (IPE) were proved to be efficient to produce gold nanoparticles (AuNPs) with uncontrollable morphologies and sizes. In order to improve the quality of the AuNPs, the IPE of P. sanguineus were successfully graded into five fractions by ultrafiltration approach, reacting with AuCl4
- respectively. Comparing with the original IPE and other fractions, proteins of molecular weight around 10–30 kDa obtained AuNPs with the most concentrated size smaller than 30 nm in high yield (above 90%) and the most homogeneous shape (87% spherical shapes). Under the optimized conditions of 25 ml 10–30 kDa dosage, pH=4, 30 ℃ and 0.5 mM AuCl4
-, the proportion of spherical AuNPs with narrower size (6–25 nm) rose up to 93.5%. The mechanism might be mainly contributed by the absorption of substrate by the abundant of amine and carboxyl functional groups, the reduction and formation of AuNPs with smaller size by larger proportion of lysine and tyrosine, and the generation of spherical products with low content of amino acids for anisotropic growth. This work provided evidence of efficient utilization of microbes in biosynthesis of precious metal nanoparticles and potential enhancement of the performance of biosynthesized products.