Earthworms play a critical role in soil ecosystems. Analyzing the spatial structure of earthworm burrows is important to understand their impact on water flow and solute transport. Existing in-situ extraction methods for earthworm burrows are time-consuming, labor-intensive and inaccurate, while CT scanning imaging is complex and expensive. The aim of this study was to quantitatively characterize structural characteristics (cross-sectional area (A), circularity (C), diameter (D), actual length (Lt), tortuosity (τ)) of anecic earthworm burrows that were open and connected at the soil surface at two sites of different tillage treatments (no-till at Lu Yuan (LY) and rotary tillage at Shang Zhuang (SZ)) by combining a new in-situ tin casting method with three-dimensional (3D) laser scanning technology. The cross-sections of anecic earthworm burrows were almost circular, and the C values were significantly negatively correlated with D and A. Statistically, there were no significant differences in the τ values (1.143 ± 0.082 vs 1.133 ± 0.108) of anecic earthworm burrows at LY and SZ, but D (6.456 ± 1.585 mm) and A (36.929 ± 21.656 mm2) of anecic earthworm burrows at LY were significantly larger than D (3.449 ± 0.531 mm) and A (9.786 ± 2.885 mm2) at SZ. Our study showed that burrow structures at two different sites differed from each other. Soil tillage methods, soil texture and soil organic matter content at the two sites could have impacted earthworm species composition, variation of earthworm size and the morphology of burrows. The method used in this research enabled us to adequately assess the spatial structure of anecic earthworm burrows in the field with a limited budget.