This study aims to establish a novel approach to better understand and predict material behavior in multi-scale lamellar microstructures. High-resolution reconstruction is built on collaborative characterization at both grain and sub-grain scale. A framework for microstructural characterization integrates, secondary electron imaging (SEI), electron backscatter diffraction (EBSD), and image recognition, to ensure that the reconstructed microstructure adequately represents the equivalent grain size, lamellar spacing, and lamellar geometric orientation. Importantly, a high-resolution reconstruction algorithm was developed combining the Laguerre-Voronoi tessellation and minimum bounding sphere (MBS), which allows the reconstructed model retain multi-scale microstructures. Finally, the mechanical properties of pearlite were investigated by combining crystal plasticity simulation with in-situ scanning electron microscopy (SEM) tensile testing based on high-resolution reconstruction. Stress-strain field analysis revealed that cross-lamellae strain bands originate from ferrite large deformation-induced shear transmission, with marked strain distribution heterogeneity within identical lamellae. This concurrently validated the excellent reliability of the novel strategy.