A novel non-contact optical inspection technique is proposed to precisely reconstruct the three-dimensional surface structure of wood and evaluate its roughness. The feasibility of using image fusion techniques to characterize the 3D roughness of wood surfaces is explored by combining high-resolution serial image acquisition techniques with advanced data processing methods. The work focuses on examining various cut surfaces of three distinct wood species: Larch, Catalpa, and Toona sinensis. The proposed 3D roughness parameters are verified using 3D reconstruction and precise measurements. The experimental results demonstrate that the proposed method exhibits strong adaptability in measuring wood surface roughness and achieves a high degree of consistency with the results obtained from traditional digital microscope systems. This study not only validates the possible use of multilayer sequence image fusion technology in measuring wood roughness, but also establishes a solid basis for the creation of 3D assessment criteria for wood surface roughness and associated inspection systems.