Considering the dependence of ε-Poly-L-lysine fermentation on antibiotics, Streptomyces diastatochromogenes (S.diastatochromogenes) expression system, which could express the ε-Poly-L-lysine stably without the addition of antibiotics, was constructed based on the type II toxin-antitoxin system relBE2sca. The antitoxin relB2sca and pls gene encoding the ε-PL synthetase were cloned into the expression vector, which was transformed into the S.diastatochromogenes without the pls gene, and the relE2sca was integrated into S. diastatochromogenes chromosome (mutant YY3), producing the mutant YY1, harboring the stable expression system of ε-Poly-L-lysine. Compared with the controls, the mutant YY1 still could synthesize the ε-Poly-L-lysine stably without the addition of antibiotic after several transmissions. Finally, the expression of RelE2sca was shown to be lethal to the common Streptomyces heterologous expression hosts, implying the potential of relBE2sca as a common genetic marker.