This study was aimed to characterize the structural and physico-mechanical properties of bio-cellulose produced through cell-free system. Fourier transform-infrared spectrum illustrated exact matching of structural peaks with microbial cellulose, used as reference. Field-emission scanning electron microscopy revealed that fibrils of bio-cellulose were thicker and more compact than microbial cellulose. The specific positions of peaks in the X-ray diffraction and nuclear magnetic resonance spectra indicated that bio-cellulose possessed cellulose II polymorphic structure. Bio-cellulose presented superior physico-mechanical properties than microbial cellulose. The water holding capacity of bio-cellulose and microbial cellulose were found to be 188.6±5.41 and 167.4±4.32 times their dry-weights, respectively. Tensile strengths and degradation temperature of bio-cellulose were 17.63MPa and 352°C, respectively compared to 14.71MPa and 327°C of microbial cellulose. Overall, the results indicated successful synthesis and superior properties of bio-cellulose that advocate its effectiveness for various applications.