Plant height is an important parameter of plant phenotype as one indicator of plant growth. In view of the complexity and scale limitation in current measurement systems, a scaleless method is proposed for the automatic measurement of plant height based on monocular computer vision. In this study, four peppers planted side by side were used as the measurement objects. Two color images of the measurement object were obtained by using a monocular camera at different shooting heights. Binary images were obtained as the images were processed by super-green grayscale and the Otsu method. The binarized images were transformed into horizontal one-dimensional data by the statistical number of vertical pixels, and the boundary points of multiple plants in the image were found and segmented into single-plant binarized images by filtering and searching for valleys. The pixel height was extracted from the segmented single plant image and the pixel displacement of the height was calculated, which was substituted into the calculation together with the reference height displacement to obtain the realistic height of the plant and complete the height measurements of multiple plants. Within the range of 2–3 m, under the light condition of 279 lx and 324 lx, this method can realize the rapid detection of multi-plant phenotypic parameters with a high precision and obtain more accurate plant height measurement results. The absolute error of plant height measurement is not more than ±10 mm, and the absolute proportion error is not more than ± 4%.