The effects of medium composition and culture conditions on the production of (6)G-fructofuranosidase with value-added astaxanthin were investigated to reduce the capital cost of neo-fructooligosaccharides (neo-FOS) production by Xanthophyllomyces dendrorhous. The sucrose and corn steep liquor (CSL) were found to be the optimal carbon source and nitrogen source, respectively. CSL and initial pH were selected as the critical factors using Plackett-Burman design. Maximum (6)G-fructofuranosidase 242.57 U/mL with 5.23 mg/L value-added astaxanthin was obtained at CSL 52.5 mL/L and pH 7.89 by central composite design. Neo-FOS yield could reach 238.12 g/L under the optimized medium conditions. Cost analysis suggested 66.3% of substrate cost was reduced compared with that before optimization. These results demonstrated that the optimized medium and culture conditions could significantly enhance the production of (6)G-fructofuranosidase with value-added astaxanthin and remarkably decrease the substrate cost, which opened up possibilities to produce neo-FOS industrially. (C) 2011 Elsevier Ltd. All rights reserved.