Printing defects are extremely common in the manufacturing industry. Although some studies have been conducted to detect printing defects, the stability and practicality of the printing defect detection has received relatively little attention. Currently, printing defect detection is susceptible to external environmental interference such as illuminance and noise, which leads to poor detection rates and poor practicality. This research develops a printing defect detection method based on scale-adaptive template matching and image alignment. Firstly, the research introduces a convolutional neural network (CNN) to adaptively extract deep feature vectors from templates and target images at a low-resolution version. Then, a feature map cross-correlation (FMCC) matching metric is proposed to measure the similarity of the feature map between the templates and target images, and the matching position is achieved by a proposed location refinement method. Finally, the matching image and the template are both sent to the image alignment module, so as to detect printing defects. The experimental results show that the accuracy of the proposed method reaches 93.62%, which can quickly and accurately find the location of the defect. Simultaneously, it is also proven that our method achieves state-of-the-art defect detection performance with strong real-time detection and anti-interference capabilities.