Automatic transplanters can improve the transplanting efficiency of vegetable seedling plugs, which are widely applied and valued in production. However, the transplantation of automatic transplanters is limited by pick-up failure or root lump destruction. To get plug seedling quality suitable for mechanical transplantation, orthogonal experiments targeting at cucumber plug seedlings and involving factors of substrate composition (ratios of sphagnum peat moss to perlite and vermiculite volume were 2∶1∶1,3∶1∶1 and 4∶1∶1), substrate compaction (ratios of substrate to pore volume were 1.0, 1.2 and 1.4) and liquid fertilizer concentration (EC values were 1.0mS/cm, 1.2mS/cm and 1.4mS/cm) were conducted. The cucumber plug seedlings were compressed or drawn by using a texture analyzer to test the compressive resistance and plug abscission force. The stem thickness, plant height, dry root weight and dry stem weight were measured, and the growing quality was evaluated using a sound seedling index. Root lumps were scanned by an X-ray micro-computed tomography (μCT). Roots were isolated from root lumps by using a threshold segmentation method and three-dimensionally reconstructed. The roots were divided into five equal parts vertically and six equal parts horizontally;the ratio of root volume in each part to the volume of root lump in this zone was defined as the root density. Then the root volume and density were statistically analyzed. It was found that the compressive resistance and plug abscission force were both significantly affected by the substrate composition and substrate compaction. Both compressive resistance and plug abscission force were improved with the rise of sphagnum peat moss concentration or compaction degree. The sound seedling index was firstly increased and then decreased with the rising concentration of sphagnum peat moss in the substrate, compaction and fertilization concentration. Vertically, the ratio of minimum to maximum root distribution densities was defined as the root uniformity degree, which was used to evaluate the root uniformity. Horizontally, the peripheral root distribution density was used to evaluate the lump enveloped degree. Clearly, substrate composition, compaction and fertilization concentration all significantly affected the root uniformity coefficient and root enveloping coefficient. Multi-object optimization of compressive resistance, plug abscission force, sound seedling index, root uniformity and peripheral root distribution density were conducted via a comprehensive scoring method, which identified the optimal three-factor combination were substrate composition of 3∶1∶1, substrate compaction of 1.2 and liquid fertilizer EC value of 1.2mS/cm. Experiments on automatic pick-up transplanter showed at the frequency of clamping seedlings were 43 plants/min, 48 plants/min and 53 plants/min, the root lump destruction rates were 3.6%, 3.9% and 4.3%, respectively, the pick-up success rates were 97.2%, 96.1% and 94.8%, respectively, which were better than other factor combinations and consistent with the comprehensive scoring result.