This article describes a comprehensive practical laboratory method for developing an enzyme to more easily measure glyphosate levels in solution. Through this article, undergraduate students of biology majors can conduct research experiments in critical fields by utilizing various techniques, such as chemiluminescence (CL) biosensors with engineered enzymes and are guided in molecular biology laboratories. A glyphosate oxidase mutant library was constructed by DNA shuffling, and a glyphosate oxidase variant with increased glyphosate degradation activity was selected by using a high-throughput screening assay. Following protein overexpression in "Escherichia coli" (DE3) and purification by affinity chromatography, the glyphosate oxidase variant protein combined with luminol-H[subscript 2]O[subscript 2] reaction was constructed as a new CL biosensor for detecting glyphosate in soils.