Currently applied methodologies (acidic or enzymatic chitosanolysis) towards synthesis of Chitosan-Oligosaccharides (COS) are faced with some drawbacks that confine their potential in biotechnological applications. The present work addresses a novel tailored method as an alternate to biosynthesize COS. An empirical statistical-mathematical approach [Plackett-Burman. Design (PBD) followed by Box-Behnken design (BBD)] was employed to optimize COS biosynthesis by an endo-chitosanase non-aflatoxigenic producing Aspergillus sp. section Flavi strain EGY1 DSM 101520 whole cells. Optimal levels localized by BBD for three key determinants were 0.687% (w/v) low molecular weight chitosan (LMWC), 25 degrees C incubation temperature and 72-96 h incubation time, along with 2.0 gL(-1) maximal level of COS, achieving 94.7% model adequacy upon growing the fungus on LMWC-based tap water medium [merely 0.687% (w/v) LMWC]. Batch mode scaling up in the laboratory scale fermenter achieved 5.34 gL(-1) of COS after 44 h with 2.67 fold enhancement. Partially acetylated hetero-oligomers with DPs (2-11) of the water-soluble COS were proved by TLC, MALDI-TOF-MS, H-1 NMR, C-13 NMR and FTIR. To the best of the authors' knowledge, the present work is the first study highlighting a novel, robust, cheap, environmentally safe, reproducible approach to biosynthesize COS by fungal whole cells.