In this study, Lactobacillus plantarum R23 was used as the tested bacteria, and the effects of collecting conditions, pre-freezing conditions and freeze-drying time on cell viability were studied by scanning electron microscope,transmission electron microscope,and the orthogonal test. The results showed that the amount of bacteria reached the maximum and higher activity at the 15th hour during the process of incubation, which might be considered as the suitable harvest time. The cell survival could reach 92.4% under optimum conditions, of which the centrifugal force played a key role. The optimum temperature and duration of gradient pre-freezing were -20, -40, -80℃ and 1,2, 3h, respectively. And the optimum moisture content of Lactobacillus plantarum R23 to be storaged was 1.20%. Based on this study, the interference mechanism of the key factors for DVS preparation on cells was explicited, and the suitable preparation conditions were obtained, which was a guidance to the large scale production of high efficient DVS.