Disclosed is a p-hydroxyphenylalanine test strip and a preparation method, comprising : a filter paper and tyrosinase and 4-aminoantipyrine loaded on the filter paper. The preparation method comprises : preparing a buffer solution, dissolving components which to be loaded on the filter paper in the buffer solution, applying a mixed solution to the filter paper by dipping or spraying, and then drying. The test strip may be used for preparing a test kit for p-hydroxyphenylalanine in urine. The test strip uses tyrosinase to detect the content of p-hydroxyphenylalanine, the components thereof are environmentally-friendly, no mercury, nickel or other heavy metal ions are contained therein, and environmental pollution will not be produced after use. In addition, the test strip is convenient to store and transport, and is convenient to use, and no waste liquid is produced.