anocellulose was produced from cotton lint and it was used as an agent of cryoprotective medium. The microstructures and surface morphologies were examined by field emission scanning electron and atomic force microscopes. Particles had a width of less than 50 nm and the length of hundreds of nanometers to several micrometers. According to the results of wide-angle X-ray diffraction, crystalline index was 74.07% and the average thickness of nanocellulose was 3.18 nm. Response surface coefficients and P values showed that the viability of Lactobacillus plantarum after freeze drying was highly dependent on nanocellulose concentration (P < 0.01). The highest viability of L. plantarum was obtained at the concentration of skim milk 13.75%, the concentration of trehalose 20.5% and the concentration of nanocellulose 13.75%. Under the optimized conditions, the predicted survival rate and observed experimental values for L. Plantarum, were 1.93 ± 0.1 × 109 CFU/ml and 1.85 ± 0.1 × 109 CFU/ml, respectively.