In order to solve the problem of handwriting identification in the field of scientific document examination in court, we usedreflectance transformation imaging to observe the three-dimensional characteristics of sister lines and quantitatively analyze them for the first time. In this experiment, the writing robot was employed to regulate the writing strength, writing angle, azimuth angle, and pen and paper clamp angle. A total of 21 common domestic pens were utilized to create “Lin” samples under the pen and paper clamp angles of 30°, 45°, and 60°. The experimental samples were produced at azimuth angles of 0°, 30°, 45°, and 60°, and at 0°, 30°, and 45° with respect to the line of sight. In total, 336 sets of samples were produced and imaged under the normal line visualization mode. The sister line characteristics and quantitative analysis were then conducted. A total of 336 sets of experimental samples were imaged and analyzed in the normal visualization mode, and the sister line characteristics of each set of samples were analyzed to derive the optimal conditions for the appearance of sister lines. Subsequently, under the optimal conditions, the factors affecting the sister lines were investigated by 3D reconstruction and quantitative analysis based on RTI technology. Finally, the probability of sister line appearance of the samples under different pen and paper clip angles was analyzed by ANOVA. Preliminary experimental results show that the sister line feature is most obvious when the azimuth angle is 45° and the pen and paper clamp angle is 45°, and the probability of the sister line appearing is as high as 99% when the experiment is repeated; the pen and paper clamp angle has a decisive influence on the formation of the sister line. The combination of RTI technology and sister lines provides new possibilities for the supplementation and enrichment of the handwriting inspection method system, and has a broad development prospect in the field of document inspection.