The critical dimension scanning electron microscope (CD-SEM) is a standard instrument for standardizing micro- to nano-sized line spacing samples. To improve the calibration accuracy of samples, this paper studies a measurement algorithm based on image processing technology. First, the characteristics of the developed samples are analyzed. Second, the algorithms for micro- to nano-sized line spacing measurement and linear approximation are researched and the line spacing standard samples with a single period from 100 nm to ~10 μm are measured. Finally, a nano-measuring machine is used in comparative experiments. The experimental results show that the relative error of the linear approximation algorithm is controlled within 0.45%. In contrast, the relative error obtained by the line spacing measurement algorithm is controlled within 0.35%, thus improving the measurement accuracy of the line spacing. The algorithm provides a measurement scheme for improving the reliability of the line spacing measurement instrument and ensuring the precision of semiconductor device manufacturing.