Paenibacillus polymyxa is a rhizobacterium that has attracted substantial attention due to its ability to produce functional metabolites and promote plant growth. Metabolic and genetic improvements in this species will benefit research and other applications of the bacterium. However, a suitable gene expression system has not been established in this species. In this study, a promoter trap system based on a green fluorescent protein and a chloramphenicol-resistance gene was developed to isolate native promoters of P. polymyxa SC2-M1 to regulate gene expression. Through high-throughput screening, the novel promoter PLH-77 was identified, sequenced, and subsequently characterized. Promoter PLH-77 is a strong, continuous expression system containing the typical -10 and -35 motifs regions. Its effective sequence was evaluated and then cascaded to improve the promotion efficiency. To further verify the existence of PLH-77, a heterogenous xylose isomerase was expressed by PLH-77 in P. polymyxa SC2-M1. In the resulting strain, the amount of xylose consumed was increased by 2.5 g/L during the 78 h fermentation period. Meanwhile, the production levels of lactate and acetate increased. It was confirmed that promoter PLH-77 could effectively mediate gene expression in P. polymyxa SC2-M1 and will further benefit the quantitative monitoring of gene expression in P. polymyxa.