NiFe-based layered double hydroxides (LDHs), as engineered nanomaterials (ENMs), are widely utilized in industrial applications, environmental remediation, and soil improvement. However, the biological impacts of such ENMs on plant-soil systems remain insufficiently explored. A 50-day soil cultivation experiment was conducted to assess the effects of two-dimensional and three-dimensional NiFe-based LDHs (2D NiFe-LDHs and 3D NiFeS-LDHs) on ryegrass growth and soil ecosystem. Generally, morphology and structure differences influence the biological effects of NiFe-based LDHs. 2D NiFe-LDHs promote ryegrass growth at specific concentrations, whereas 3D NiFeS-LDHs exhibit significant inhibitory effects on ryegrass growth. NiFe-based LDHs directly impacted soil geochemistry. 3D NiFeS-LDHs decreased soil pH while increasing electrical conductivity and soil organic carbon. 2D NiFe-LDHs inhibited soil sucrase (S-SC) activity, but enhanced soil urease (S-UE) activity. Conversely, NiFeS-LDHs inhibited catalase (S-CAT) activity and S-UE activity, while promoting neutral phosphatase (S-NP) activity However, as concentrations increased, the effects of 2D NiFe-LDHs and 3D NiFeS-LDHs on soil properties and enzyme activities became more similar. Both NiFe-based LDHs altered the diversity of soil bacterial and fungal communities, with 3D NiFeS-LDHs exerting a stronger inhibitory effect on fungal diversity at higher concentrations. The structural differences in NiFe-based LDHs exert a distinct and substantial impact on the composition of soil microbial communities and soil enzyme activities. The findings provide critical insights into bio-effects of NiFe-based LDHs, which provide a theoretical basis for the study of potential ecological risks in soil.