The boundary slip length at solid-liquid interfaces has significant application potential in microfluidic and nanofluidic systems. Due to their geometric universality and ease of precise control, cylindrical micro/nano structures are widely used in micromanufacturing technologies. Existing research indicates that cylindrical microstructures are more prone to boundary slip compared to other structures. To reveal the mechanism of how the size and spacing of cylindrical micro/nano structures affect slip length, this study utilizes COMSOL simulation to analyze the effects of cylindrical microstructure size and spacing on flow rate. Flow rate data under various parameters were obtained and analyzed to determine that the cylindrical microstructure size has a minor impact on boundary slip length, which decreases initially with increasing size and then stabilizes. In contrast, the spacing of cylindrical microstructures has a significant effect on boundary slip length; as the spacing increases, the boundary slip length shows a positive correlation with cylindrical model spacing from 1 to 12 μm, peaking at 12 μm, and then gradually stabilizing.Colloidal probe contact mode AFM (CM-AFM) was used to measure the boundary slip length of deionized water at room temperature and pressure under different surface microstructure sizes and spacings. For a constant microstructure spacing, the boundary slip lengths of samples with cylindrical diameters of 3 μm, 4 μm, 5 μm, and 6 μm were 2574 nm, 3206 nm, 3441 nm, and 3791 nm, respectively. For cylindrical spacings of 2 μm, 3 μm, and 4 μm, the boundary slip lengths were 2574 nm, 2786 nm, and 3449 nm, respectively. The experimental results show that both the size and spacing of cylindrical microstructures affect the boundary slip length. At the microscopic scale, larger cylindrical microstructure sizes and spacings tend to form nanoscale bubbles between structures, reducing fluid resistance on the solid surface and increasing boundary slip with increasing size and spacing. Comparison of simulation and experimental results indicates that in microchannel design, altering the surface size and spacing can reduce fluid resistance. By optimizing these parameters, effective control of boundary slip length can be achieved, thereby enhancing the performance and efficiency of microfluidic systems.