Abstract: Symbiotic nitrogen fixation of soybean-rhizobium combinations is an essential biological pathway to convert atmospheric nitrogen into plant-available nitrogen nutrients. Both nitrogen supply and crop productivity are also improved in the maize-soybean intercropping system. However, conventional destructive sampling cannot continuously observe underground root growth and nodule dynamics. It is also unclear about the regulatory relationships between soybean genotype-rhizobium matching, root spatial interaction, and the efficiency of symbiotic nitrogen fixation. Therefore, it is required to precisely regulate high-efficiency nitrogen utilization in intercropping agroecosystems. In this study, a systematic investigation was conducted to clarify root spatial distribution, root length density, and nodule development in maize-soybean intercropping. A modular rhizobox was also utilized with in situ monitoring of root morphology. Nitrogen fixation performance and yield formation were determined under different combinations of soybean-rhizobium intercropping. The results showed that rhizobium inoculation significantly altered the directional growth characteristics of maize roots towards intercropped soybean roots. The Huai11-Sr-Su29 (Huaidou11-Sinorhizobium fredii-Suyu29) treatment exhibited an outstanding trend of maize roots proliferating toward soybean root zones, thus forming intensive root interaction regions. Both Tai6-Bj-Su29 (Taifeng6-Bradyrhizobium japonicum-Suyu29) and Tai6-Sr-Su29 (Taifeng6-Sinorhizobium fredii-Suyu29) treatments shared only slight directional growth of maize roots toward soybean roots. In contrast, the Huai11-Bj-Su29 (Huaidou11-Bradyrhizobium japonicum-Suyu29) treatment presented a root spatial distribution similar to that of the non-inoculated group. There was only a small root overlap and no outstanding cross-interaction. The root length density was lower than 0.2 cm/cm³ in the interaction zone. Symbiotic matching analysis indicated that the two rhizobial strains presented host genotype specificities. Bradyrhizobium japonicum (Bj) displayed excellent cultivar matching specificity, while Sinorhizobium fredii (Sr) had broader symbiotic adaptability to form effective nodules with both soybean cultivars. There were only a few differences in nodulation initiation time among treatments. In Tai6 (Taifeng 6), an efficient symbiosis relationship was obtained to form nodules with the rhizobial strains. Whereas in Huai11 (Huaidou 11), high-efficiency nodulation was achieved using the Sr strain, indicating symbiotic performance with the Bj strain, and the maximum nitrogenase activity of 85.46 μL/(g·h). Among all treatments, the Tai6-Sr-Su29 combination exhibited optimal symbiotic performance, with early nodule development, the maximum nodule diameter of 7 mm, and the highest nitrogenase activity of 223.2 μL/(g·h). Interlaced but non-entangled root spatial structures were observed to effectively alleviate interspecific root competition, leading to high maize and soybean yield. Correlation analysis revealed that both nitrogenase activity and single-plant nitrogen fixation potential were extremely significantly and positively linearly correlated with nodule diameter (R²>0.92, P<0.01). Rhizobium inoculation significantly regulated the biomass allocation of the intercropping system. Compared with the non-inoculated group, inoculated soybean reduced root-shoot ratio (P<0.01) by 20%-30%, whereas there was an increase in grain yield (P<0.01) of 30%-40%. Rhizobium inoculation reduced redundant biomass allocation to soybean roots, thus promoting the translocation and accumulation of photosynthates in grains, particularly for the high crop yield. There were genotype-specific symbiotic matching and root spatial interaction of soybean-rhizobium in the soybean-maize intercropping system. Nodule diameter was used as a reliable visual indicator for rapid and non-destructive evaluation of symbiotic nitrogen fixation capacity. The findings can provide a theoretical basis and technical reference to optimize rhizobium inoculation for nitrogen use efficiency and productivity in intercropping farmland.