Disclosed is a lyophilized powder for detecting p-hydroxyphenylalanine, a preparation method, and an application thereof. The lyophilized powder contains tyrosinase and 4-aminoantipyrine. The preparation method includes preparing a buffer, dissolving other components in the buffer, lyophilizing the mixed solution at -80癈, and drying the same with a freeze dryer. The lyophilized powder can be used to prepare a testing kit for detecting p-hydroxyphenylalanine in urine. The technical solution uses tyrosinase to detect the concentration of p-hydroxyphenylalanine, and successfully develops the same to obtain a lyophilized powder product for urine testing. The test reagent components are environmentally friendly, free of heavy metal ions such as mercury or nickel, and no environmental pollution will be caused after use. In addition, the lyophilized powder is convenient for storage and transportation, and is also convenient to use.