The invention provides preparation and application of an immunochromatography method and test paper for quantitative detection of prealbumin. The test paper consists of a bottom plate, a sample pad, a conjugate pad, a nitrocellulose membrane and a piece of water absorbing paper, wherein the sample pad, the conjugate pad, the nitrocellulose membrane and the water absorbing paper are sequentially adhered to the bottom plate in a lap joint manner and are respectively and partially overlapped; a detection line and a quality control line are arranged on the nitrocellulose membrane; the quality control line is wrapped with a donkey-anti-rat antibody. A method comprises the following steps: (1) adding a solution which is of known concentration and comprises a prealbumin standard substance onto the sample pad of the test paper, and drawing a standard curve; (2) adding a solution of a substance to be tested, performing reaction, and reading content according to standard curves. The test paper and the detection method provided by the invention are high in sensitivity, that is, up to pg / mL, wide in detection linearity, convenient to operate, and short in time, that is, results can be obtained within only 10-20 minutes.