The invention discloses a polyolefin nanometer fiber film sensor for rapid bacterium detection and a preparation method thereof. According to the invention, polyolefin copolymer is adopted as a substrate material, and the chemically-modified polyolefin nanometer fiber film biosensor is prepared by surface chemical modification, and solid phase synthetic process of bioactive molecules. The nanometer fiber film biosensor prepared by the method of the invention overcomes, with respect to performance, the disadvantages of time consumption, manpower consumption, poor accuracy for bacterium culture and bacterial colony counting in traditional bacterium detection, realizes rapid detection of ATP released by bacteria by using the luminescence principle between triphosadenine, luciferase, and fluorescein, and realizes rapid response to biological harmful substances. With the solid phase synthesis, bioactive enzymes are well fixed on the surface of the nanometer fiber film, which realizes repeated and circular detection of bacteria. The nanometer fiber film sensor is widely applicable to fields of biotechnology, medical health, food and medicine detection, water treatment, and the like.