[Background] Mangrove habitats are rich in microbial resources. Isolation of more pure culture actinomycetes can provide strain resources for the discovery of new antibiotics. [Objective] A new actinomycete isolation medium was used to explore the strain resources of actinomycetes in mangrove habitats of Maowei Sea in Guangxi Beibu Gulf. Separation effect of new actinomycetes isolated culture medium was evaluated. [Methods] Coconut juice-mycose medium was newly designed and used for isolating actinobacteria from mangrove rhizosphere soil and mangrove root bark in Guangxi Beibu Gulf, Maowei Sea. The diversity analysis of pure culture actinomycetes was carried out based on molecular biology method. [Results] 65 strains of actinomycetes, including 4 potential new taxa, were isolated from the rhizosphere soil of mangrove which distributed in 5 orders, 10 families and 15 genera. Seven, five and eleven genera were isolated by YIM171, MA and coconut juice-mycose medium respectively. 19 strains of actinomycetes, including 4 potential new taxa, isolated from the root bark of mangrove were distributed in 4 orders, 7 families and 10 genera, 7 genera were isolated by YIM171 medium and coconut juice-mycose medium, respectively. Three potential new taxa were obtained from two kinds of samples in mangrove habitats using Coconut juice-mycose medium. [Conclusion] Rhizosphere soil and root bark of mangrove in Guangxi Beibu Gulf, Maowei Sea present an extremely abundant habitats for the isolation of a significant diversity of actinomycetes. Coconut juice-mycose medium is effective in the isolation of actinomycetes thus can be considered as the isolation medium for actinomycetes.