A facile one-pot eco-friendly process for synthesis of gold nanoparticles (AuNPs) with high catalytic activity was achieved using cell-free extracts of Aspergillus sp. WL-Au as reducing, capping and stabilizing agents. The surface plasmon resonance band of UV-vis spectrum at 532 nm confirmed the presence of AuNPs. Transmission electron microscopy images showed that quite uniform spherical AuNPs were synthesized and the average size of nanoparticles increased from 4 nm to 29 nm with reaction time. X-ray diffraction analysis verified the formation of nano-crystalline gold particles. Fourier transform infrared spectra showed the presence of functional groups on the surface of biosynthesized AuNPs, such as O-H, N-H, C=O, C-H, C-OH and C-O-C groups, which increased the stability of AuNPs. The biogenic AuNPs could serve as a highly efficient catalyst for 4-nitrophenol reduction. The reaction rate constant was linearly correlated with the concentration of AuNPs, which increased from 0.59 min(-1) to 1.51 min(-1) with the amount of AuNPs increasing form 1.46 x 10(-6) to 17.47 x 10(-6) mmol. Moreover, the as-synthesized AuNPs exhibited a remarkable normalized catalytic activity (4.04 x 10(5) min-1 mol(-1)), which was much higher than that observed for AuNPs synthesized by other biological and conventional chemical methods. (C) 2016 Elsevier B.V. All rights reserved.