Accurate identification of Hepatitis B virus (HBV) disease by analyzing the Raman spectroscopic images is a challenge for pathologists. To save precious human lives, an efficient technique is required with higher diagnostic accuracy at early‐stage of HBV. We proposed a novel method of HBV diagnosis using deep neural networks with the concept of transfer learning and Raman spectroscopic images. The proposed approach developed by utilizing pretrained convolutional neural networks ResNet101 by employing transfer learning on a real dataset of HBV‐infected blood plasma samples. Dataset consists of 1000 Raman images in which 600 are HBV‐infected blood plasma samples, and 400 are healthy ones. The developed model is capable to detect minute variation between infected and healthy samples and achieved enhanced performance. The proposed approach has been assessed and attained high classification accuracy, sensitivity, specificity, and AUC of 99.7%, 100%, 99.25%, and 98.7%, respectively. The proposed TL‐ResNet101 model outperformed the conventional approaches such as PCA‐SVM and PCA‐LDA and demonstrated improved accuracy more than 7%. High performance indicates that the developed TL‐ResNet101 model has potential to use for HBV diagnosis. Moreover, the developed automated approach can be extended for other disease.