As the floral emblem of Qingdao，Camellia japonica（Naidong） is a precious ornamental species during the winter of the north， but the quantity of C. japonica（Naidong） cultivars cannot meet requirements for application in landscape at present， so it is necessary to enlarge propagation by the means of tissue culture. In order to establish the regeneration system of C. japonica（Naidong）， different factors affected on callus induction and plantlet regeneration were studied from mature cotyledons of C. japonica （Naidong）. The optimum timing of sterilization in C. japonica（Naidong） seeds was soaked for 8 min in the 2% NaClo， callus induction rate was 100%. The optimal hormone combination of cotyledon callus subculture was 0.5 mg·L-1 NAA+1.5 mg·L-1 6-BA， the multiplication rate was 4.06. After 2-time subcultured， the callus was transferred to MS+1.0mg·L-1 NAA+10.0 mg·L-1 6-BA for differentiation of adventitious buds， the rate of adventitious bud differentiation was 10.7%. The shoot base was immersed in 500 mg·L-1 IBA solution for 70 min， then transferred to 1/2MS， the rate of rooting could reach 40%. Regeneration system from mature cotyledons of C.japonica（Naidong） by inducing callus， adventitiousshoots and roots was established， which would provide technical support for the vegetative production and innovation of its germplasm resources.