There have been significant advances in literature mining, allowing for the extraction of target information from the literature. However, biological literature often includes biological pathway images that are difficult to extract in an easily editable format. To address this challenge, this study aims to develop a machine learning framework called the “Extraction of Biological Pathway Information” (EBPI). The framework automates the search for relevant publications, extracts biological pathway information from images within the literature, including genes, enzymes, and metabolites, and generates the output in a tabular format. For this, this framework determines the direction of biochemical reactions, and detects and classifies texts within biological pathway images. Performance of EBPI was evaluated by comparing the extracted pathway information with manually curated pathway maps. EBPI will be useful for extracting biological pathway information from the literature in a high-throughput manner, and can be used for pathway studies, including metabolic engineering.