The invention discloses an aerobic and bacterial vaginosis combined determination kit and a preparation method thereof. The kit comprises a color-developing agent and a reaction device, wherein the reaction device comprises a hydrogen oxide reaction pad, a neuraminidase reaction pad, a leucocyte esterase reaction pad, a beta-glucuronidase reaction pad and a coagulase reaction pad. Sample liquid is sucked by using straw, one drop of the sample liquid is dropwise added on each of the hydrogen oxide reaction pad, the neuraminidase reaction pad, the leucocyte esterase reaction pad, the beta-glucuronidase reaction pad and the coagulase reaction pad of the reaction device, the volume of each drop of the sample liquid is 30 mu l, after the reaction device is put into a water bath at a temperature of 36-38 DEG C or a dry bath at a temperature of 48 DEG C to carry out color developing for 15 minutes, one drop of color-developing liquid A is added on the neuraminidase reaction pad, one drop of color-developing liquid B is added on the coagulase reaction pad, and 30 seconds later, a result is shown. Thus, an effect of simultaneously detecting hydrogen oxide, neuraminidase, leucocyte esterase, beta-glucuronidase and coagulase to diagnose aerobic and bacterial vaginosis can be achieved.