The invention provides an AV/BV joint detection kit, comprising a reactor, sample diluent, a color-developing agent A and a color-developing agent B, wherein the reactor is provided with a hydrogen peroxide reaction hole, a neuraminidase reaction hole, a leukocyte esterase reaction hole, a beta-glucuronidase reaction hole and a coagulase reaction hole, and each reaction hole has a corresponding reaction pad. The invention also provides a preparation method of the kit. The detection kit in the invention directly identifies floras such as aerobic bacteria/anaerobic bacteria, facultative anaerobe and the like in vaginal discharge by determining hydrogen peroxide and determining the activity of neuraminidase, leukocyte esterase, beta-glucuronidase and coagulase. The method has simple, convenient and rapid operation and high accuracy, and is suitable for convention application in clinics especially in hospital outpatient.