Disclosed are a mercury-free p-hydroxyl phenylalanine detection reagent and a preparation method and an application thereof. The reagent comprises a buffer solution and tyrosinase and 4-amino antipyrine which are dispersed in the buffer solution, and may be applied to prepare a kit for p-hydroxyl phenylalanine in urea. The preparation method is to meter and mix the above components. Tyrosinase is used for qualitatively and quantitatively analyzing the p-hydroxyl phenylalanine, the p-hydroxyl phenylalanine is successfully improved into a urine detection reagent that can be produced, the components of the detection reagent are environment-friendly, the detection reagent contains no heavy metal ions of mercury ions, nickel ions, and the like any more, and environment pollution cannot be caused after use; in addition, a dispersion system is gentle, so that preparation and use processes are quite safe.