The abundance of microplastics (MPs) in soil environments has attracted significant attentions, due to their impact on soil physico-chemical properties. However, limited information is available on the influences of MPs on soil carbon composition and microbial utilization characteristics. Therefore, a two-month incubation experiment was conducted to add polyethylene microplastics (PE-MPs) with different levels (1%, 10%) and sizes (150–300 μm and 75–150 μm) into different soils. After that, soil chemical properties including the dissolved organic carbon (DOC), spectral characteristics of dissolved organic matter (DOM) and soil microbial characteristics were analyzed. Results revealed that PE-MPs addition caused significant differences in soil chemical properties between farmland and woodland soils, particularly in soil pH, DOM composition, and soil phosphatase activity. Woodland soil always exhibited higher levels of DOC content, microbial diversity, and soil carbon source utilization compared to farmland soil, leading to increased humification in the DOM of woodland soil. PE-MPs with a larger particle size significantly increased both the soil DOC content and enzyme activity. Addition of PE-MPs altered the soil DOM composition, and the fluorescence parameters like the biological index (BIX) and humification degree. Moreover, the carbon source utilization intensity of microorganisms on PE MPs-contaminated soils is higher in woodland soils. Various analyses confirmed that compared to other soil properties, characteristics of soil DOM had a more significant impact on soil microbial community composition. Thus, PE-MPs in conjunction with soil DOM spectral characteristics regulated soil microbial diversity, which is crucial for understanding soil carbon sequestration. Display omitted •Significant differences in DOM composition between woodland and farmland soils.•PE addition increased the carbon utilization intensity of soil microorganisms.•PE size coupled with DOM spectral characteristic regulated the soil microbial diversity.