The invention discloses a rapid nucleic acid test strip detection kit for a food-borne pathogenic microorganism cronobacter sakazakii and an application method of the kit, and belongs to the fields of molecular biology and immunology. According to the kit and the application method thereof, a high-sensitivity and high-specificity polymerase chain reaction method for nucleic acid detection is combined with an immune colloidal gold rapid-detection technology for immunological detection, unique primers are designed and labeled, extracted target DNA (deoxyribonucleic acid) is subjected to specific amplification, and an amplification product is bound with a gold labeled antibody fixed on a test strip in a developing solution to form a stable and visible detection strip and a stable and visible quality control strip, so that main food-borne pathogenic microorganisms are rapidly and accurately detected.