Cholesterol esterase from Pseudomonas fluorescens is widely used in clinical diagnostics for the enzymatic determination of cholesterol in serum. It specifically hydrolyzes cholesterol esters to free cholesterol and free fatty acids, enabling accurate measurement of total cholesterol levels when combined with other enzymes like cholesterol oxidase. This reaction is critical in automated clinical analyzers and diagnostic kits.

The enzyme is favored in diagnostic applications due to its high specificity, stability, and activity under assay conditions. It plays a key role in lipid profile assessments, helping evaluate cardiovascular disease risk. Additionally, it is used in research settings to study lipid metabolism and enzyme kinetics. Its bacterial origin allows for consistent production and scalability in industrial applications.