Tapping scanning mode is an important method for measuring surface topography at the nanometer scale. It is widely used because it can eliminate lateral force and reduce damage to the tested sample. Research on three dimensional (3D) scanning technology with a large range and high depth-to-width ratio has important practical significance and engineering application value because the current scanning probe microscope has the limitations of small measurement ranges and weak Z-direction measurement ability. The high-frequency resonance of the quartz tuning fork, combined with the tungsten stylus, is used in this paper. It has the ability to measure the surface profile of the microdevice with a large aspect ratio. The proposed 3D scanning measurement system has realized a microstructure measurement with a depth of ∼58 µm. The entire measuring range is 400 × 400 × 400 µm3, and the vertical resolution reaches 0.28 nm. The system can accurately obtain the 3D surface topography of the microfluidic biochip. In addition, a sliding window algorithm (SWA) based on errors in the scanning process and low scanning efficiency is proposed. Compared with the point-by-line scanning algorithm, the proposed SWA reduces the mean value of the squared residuals of the 3D profile by 7.70%, thereby verifying the feasibility of the algorithm. The 3D scanning measurement system and the algorithm in the tap mode provide an important reference for the 3D topography measurement of microstructures with large aspect ratios.