Abstract: Ultra-fine positioning stages are the indispensable components in many areas of nanotechnology and advanced material analysis, and are always integrated into analytical devices such as Scanning Probe Microscope (SPM), optical microscope. The mechanical properties of the microscopic measurement system were strongly influenced by the nano-mechanical performance of an ultra-fine positioning stage. A traceable calibration setup for investigating the quasi-static performance of nano-positioning stage was developed, which utilized a differential plane mirror interferometer with double-pass configuration from the National Physical Laboratory (NPL). Based on an NPL-developed FPGA and LabView, the laser interferometric data acquisition (DAQ) and data decoding system with high precision and stable frequency was built up to enable traceable quasi-static calibration of ultra-fine nano positioning stages. Furtherly, the proposed system was used to calibrate and analyze the metrological characteristics of nano-positioning stages. The experimental results have proven that the calibration setup can achieve a noise floor lower than 10 \rmpm/\sqrt \rmHz under nearly open-air conditions. The calibrated pico-positioning stage has an excellent nano-mechanical performances, such as the linearity of being lower than 1.2×10⁻⁶, the resolution of being up to 40 picometer, good repeatability and stabilization. The results indicate that the proposed method and system can be used to measure the performances of the ultra-fine positioning stages, and furtherly be used for pico-indentation with indentation depths down to a few picometers and the large-scope measurement at the atomic scale.