The enzyme-linked immunosorbent assay (ELISA) is a cornerstone technique for quantifying protein secretion in biological research. However, the built-in software provided by ELISA plate readers often struggles to accurately detect low-concentration proteins, particularly in the sub-nanogram/mL range, due to limitations in calibration curve fitting. We developed the ELISA Protein Detector (EPD) to overcome these challenges. This open-source Python-based software employs advanced optimization algorithms to enhance curve fitting precision, particularly at low detection thresholds." EPD features an intuitive user interface, requires minimal technical expertise, and supports robust cross-validation to enhance the reliability of ELISA data analysis. Tested on Windows systems, this tool provides a cost-effective and versatile solution for researchers, enabling accurate quantification of low-level protein concentrations and addressing the shortcomings of standard ELISA software in diverse biological and clinical applications.