The invention discloses an explosive molecular biosensor synthesized by utilizing a regulatory element as well as a preparation method and application of the explosive molecular biosensor. The explosive molecular biosensor comprises a report element of a self-luminous operon luxABCDE operon, a sensing element of a promoter nahR, RsrR regulatory protein and a tetH promoter regulated by the RsrR regulatory protein at the downstream, according to the explosive molecule biosensor, a signal of a nahR promoter for sensing explosive molecules is subjected to cascade amplification through the RsrR-PtetH regulation and control element, so that a self-luminous signal generated after sensing the explosive molecules is enhanced, and the detection sensitivity is improved; the method and the biosensor for sensing molecular signals of explosives by regulating the protein regulation element cascade amplification promoter are established, and the sensor is wide in detection range, high in sensitivity, simple and convenient in method, low in cost and high in safety, and can be applied to the fields of environmental protection, national safety maintenance and the like.