This study attempted to explore the suitable liquid nitrogen cryopreservation condition of Murraya exotica L. seed and provided a pipeline for the long-term preservation of the seeds. Using mature seeds of Murraya exotica L. with different water content using rapid drying method, cryopreservation experiments were carried out with three approaches including vitrification method, step freezing and direct freezing method, followed by an observation of the micro-characters with the method of paraffin section and the quantification of the physiological and biochemical activity indexes. The results showed that the water content of the seed decreased from 30% to 7%, and the viability of the control group slowly decreased from 98% to 82%. After being treated by different freezing methods, the viability first decreased and then rised with the decrease of water content. At 7% water content, the viability of each freezing group reached to the highest, and the viability is the closest before and after cryopreservation at this time. The seed vigor after step freezing was generally higher than that of vitrification and direct freezing. Among them, the seed color at the cutting surface were darker than other groups after step freezing, and the seeds with 30% water content had cavity or severe plasmolysis in some cells after step freezing. The vigor and structure of the seeds in the 7% water content group were basically unchanged after the different freezing treatments compared with the control group. After the step freezing treatment, the malondialdehyde content, protein content, catalase activity and peroxidase activity of the seeds decreased and the dehydrogenase activity was found to be elevated. Thus, we concluded that the Murraya exotica L. seeds are able to be stored following the ultralow temperature preservation pipeline in liquid nitrogen, by use of the water content of 7% seed with the method of step freezing.