The engine is primary power source of harvester, and its punch combination position has a direct impact on machine assembly quality and further affections on operating efficiency and reliability of the whole machine. At present, the engine punch combination position measurement is mainly performed manually with quite low precision and bad consistency. Focused on the automatic measurement of engine punch combination position, a machine vision based online measurement method for engine punch combination position of large-scale harvesters was proposed in this paper through establishing a punch combination position error model. A certain number of cameras were used to capture the two-dimensional images of each energy mounting hole, and the punch combination position error was measured and calculated real-timely through online calibration, image enhancement, feature extraction, coordinate transformation and so on. On this basis, a measurement and control software was developed based on Labwindows/CVI, and realized the rapid measurement of punch combination position. In this paper, engine punch combination position of a certain type of harvesting chassis of corn harvester was studied. The experiment results showed that the punch combination position relationships could be achieved effectively, and the error analysis and assessment could be fulfilled by the established punch combination position error model. Furthermore, its measuring results were superior to traditional calipers measuring tools readings in resolution and accuracy with a highly efficiency. This proposed online measurement method could meet the demand of automated production-line measurements.