Quantification of legumes biological nitrogen fixation (BNF) is normally done via analytical methods that require sampling, drying, grinding, and laboratory processing. These methods are time-consuming, expensive, and are not accessible to growers. The correlation between the BNF quantity and nodule number and nodule mass can be used to develop tools that allow rapid assessments of the BNF. In this work, we developed a graphical user interface (GUI) based deep learning and image processing system for legume nodule segmentation and classification to determine the characteristics associated with legume nodules using digital images. During image acquisition, the legume root samples were imaged using a smartphone camera and lab-made imaging setup. A total of 1468 digital images were collected from 367 root systems. After the first run of imaging, nodules were separated from the roots, and another image was obtained from the nodules of each sample. For comparison and validations, nodules were manually counted, dried, and weighed. In this study, a categorized image data library was developed and utilized for deep learning and image processing. Digital image processing filters, and image segmentation method were applied to process the digital images of the root systems and determine the number of nodules and provide their characteristics. Deep learning models were used to classify the images into different legume classes. Furthermore, a GUI was successfully developed to simplify the utilization and application of deep learning/digital image processing algorithms. The preliminary results of this study demonstrate that our deep learning/image analysis system has a great potential to accurately quantify, characterize, and count the nodules and that could be extremely valuable to growers.