The invention belongs to the field of high polymer materials and biological medicines, and relates to a ferroferric oxide loaded nickel carbide layer nanotube and a preparation method and applicationthereof. The ferroferric oxide loaded nickel carbide layer nanotube (Fe3O4@C-NT / Ni) is in the shape of a hollow tube, has excellent electromagnetism, catalytic activity, biological compatibility and water dispersibility, has activity similar to that of peroxidase, and is used for detecting and analyzing hydrogen peroxide and bio-active substances. The preparation method of Fe3O4@C-NT / Ni is simpleand efficient, high in recycling rate, safe and environmentally friendly. The catalytic activity of Fe3O4@C-NT / Ni is similar to that of peroxidase, Fe3O4@C-NT / Ni is used for redox reaction of hydrogenperoxide as an electron acceptor, and qualitative and quantitative measurement of the hydrogen peroxide is realized; and Fe3O4@C-NT / Ni is also used for qualitative and quantitative measurement of bio-active substances which take the hydrogen peroxide as a constant-ratio product. Compared with the prior art, the method is simple and speedy, economical and environmentally friendly, wide in detection line, high in sensitivity, good in selectivity and visual.