The non-orthogonal shaft laser theodolite (N-theodolite) measurement system is a new kind of instrument applied to large-scale metrology. The system consists of two identical N-theodolites, each of which is made up of a two-dimensional rotary table and one collimated laser. The three axes of the N-theodolite have no strict requirements on their orthogonality and intersection conditions. A novel method, to the best of our knowledge, is proposed to calibrate the N-theodolite measurement system, and the calibration method of the laser beam for N-theodolite is the key innovation aspect of this paper. The laser beam is calibrated through dynamic rotation. Throughout the experiment, the straightness accuracy of the laser beam of the left N-theodolite is 0.08 mm and the right N-theodolite is 0.074 mm. The repeatability of the straightness accuracy is ${\pm 0.032}\,\,{\rm mm}$±0.032mm. The uncertainty of straightness accuracy is 0.040 mm. The measurement experiment results show that the maximum deviation of the measured value of this system is 0.34 mm at a range of 5 m, which is verified to be a feasible method for the calibration of the laser beam.