This research work aims to implement an automated segmentation process to extract the endoplasmic reticulum (ER) network in fluorescence microscopy images (FMI) using pretrained convolutional neural network (CNN). The threshold level of the raw FMT is complex, and extraction of the ER network is a challenging task. Hence, an image conversion procedure is initially employed to reduce its complexity. This work employed the pretrained CNN schemes, such as VGG-UNet and VGG-SegNet, to mine the ER network from the chosen FMI test images. The proposed ER segmentation pipeline consists of the following phases; (i) clinical image collection, 16-bit to 8-bit conversion and resizing; (ii) implementation of pretrained VGG-UNet and VGG-SegNet; (iii) extraction of the binary form of ER network; (iv) comparing the mined ER with ground-truth; and (v) computation of image measures and validation. The considered FMI dataset consists of 223 test images, and image augmentation is then implemented to increase these images. The result of this scheme is then confirmed against other CNN methods, such as U-Net, SegNet, and Res-UNet. The experimental outcome confirms a segmentation accuracy of >98% with VGG-UNet and VGG-SegNet. The results of this research authenticate that the proposed pipeline can be considered to examine the clinical-grade FMI.