Biological synthesis of nanomaterials using microorganisms is a growing field of industrial microbiology due to the numerous beneficial properties while lower toxicity for biological systems. We have developed a cell-free viable approach for synthesis of gold nanoparticles using alpha-NADPH-dependent sulfite reductase purified from Escherichia colt. The enzyme was purified by ion exchange chromatography on DEAE Sephadex A-50 and its molecular weight was determined by gel filtration on Sephacryl S-300. The purified enzyme with molecular weight of 116 kDa composed of two subunits of 75 and 41 kDa was successfully used for cell-free synthesis of gold nanoparticles which were spherical with an average size of 10 nm and a zeta potential of -30 +/- 0.2. Gold nanoparticles showed strong antifungal activity toward a wide range of human pathogenic fungi. Evaluation of the in vitro cytotoxicity of gold nanoparticles showed no toxicity for two cell lines, i.e., Vero and Hep-2 at the concentrations ranged from 0.31 to 10%. Taken together, biologically active non-toxic gold nanoparticles green synthesized by a sulfite reductase of E. coil in the present work are potential candidates for application in different disciplines of medicine and biology from diagnostics to drug delivery systems, combating infectious diseases and anticancer therapies. (C) 2015 Elsevier Ltd. All rights reserved.