The invention provides a conjugate of a polymer and a biological stain and a preparation method and application of the conjugate. The conjugate has the formula G-X-B, wherein the polymer G is selected from the group consisting of glucans, fructosans, polymannuronic acid, polyguluronic acid, heteropolysaccharides, mono- or hetero-polysaccharides, mono- or hetero-polyuronic acid polysaccharides, mono- or hetero-polyuronic acid sulfate esters, hydrophilic alcohol high polymers, polylysine, polyglutamic acid and polyaspartic acid; the linking group X is a C1-C10 organic group; and the biological stain or a derivative B thereof may be toluidine blue, methylene blue, Evans blue, neutral red, Janus green or bright cresol purple. The conjugate can stain bilateral neck and submaxillary or submental lymphatic vessels and lymph nodes in New Zealand white rabbits by indirect lymph staining method, without diffusion in tongue body. No obvious toxic and adverse effects appear in all experimental animals in each group, and no obvious pathological changes appear in histological examination of internal organs.