[Background] Resuscitation promoting factor (RPF) is an active protein secreted by some different actinobacteria species, which was first reported in Micrococcus luteus. It resuscitates the dormant bacteria and promotes the growth of active bacteria. However, few studies have investigated its contribution in environmental samples to the growth or resuscitation of bacteria other than the original host. [Objective] In order to find novel strains and explore the functional bacteria, we investigated the role of RPF in promoting the latent dormant bacteria from specific environmental sample. [Methods] Firstly, bioinformatics analysis and homology modeling of Micrococcus luteus RPF (MlRPF) was performed. Subsequently, the MlRPF encoding gene was cloned and expressed in Escherichia coli; Finally, we used the purified recombinant MlRPF to study the resuscitation effect toward latent dormant bacteria from an activated sludge sample. [Results] Bioinformatics analysis revealed that the MlRPF branched separately and was distant from other RPF family members in the phylogenetic tree. Structural analysis of its “RPF-domain” suggested that MlRPF has a similar catalytic structure domain as C-type lysozyme, this finding helps explain why RPF family members have lysozyme-like activity; the recombinant MlRPF was expressed in soluble form in E. coli expression system, could promote the growth of M. luteus IAM 14879 and retain its physiological activity. By used purified recombinant MlRPF, we isolated Dietzia, Paracoccus, Rhodococcus, and Brevundimonas strains from an activated sludge sample. [Conclusion] These results not only provide a novel method for the study of microbial diversity and the exploration of special microbial resources in environmental samples, but also provide a theoretical basis for the research and development of wastewater bioaugmentation technology based on MlRPF.