The mice spleens isolated from cortisol-BSA-immunized mice are fused with myeloma cells to generate hybridomas. Hybridoma cells are further injected into the abdominal cavity of mice, producing and obtaining mouse ascites fluid containing cortisol monoclonal antibody. This monoclonal antibody occurs as an unconjugated IgG1. Its purity is greater than 95% using protein G purified. The target cortisol is a glucocorticoid produced from cholesterol in the two adrenal glands located on top of each kidney. The cortisol exerts diverse functions in the mediation of the stress response, and modulation of metabolism, inflammation, and immune response. And this anti-cortisol monoclonal antibody has been validated for use in the ELISA assay.