Screen printing has been adopted for fabricating a wide variety of electronic devices. However, the printing defects and reliability have been an obstacle for industrialization of printed electronics. In this research, the artificial intelligence (AI) model was developed and integrated with the in-house roll-to-roll screen printing system to detect smearing defect, which is one of the main defects of screen printing. The U-Net architecture was adopted, and a total of 19 models were designed with model sizes ranging from 8E + 3 to 3E + 7 number of parameters. Their performances as validation mean Intersection over Union (IoU) were analyzed, and the optimal model was chosen with a validation mean IoU of 95.1% and a number of parameters of 8E + 6. The printed line images were evaluated by the AI model for various printing conditions, such as printed line widths, printing paste premixing, printing speeds, and printed line directions, which showed that the model could effectively detect the smearing defects. Also, the AI model capabilities were investigated for repeated printing, which demonstrated that it can be used for the reliability assessment of the screen printing process.