An enhancing reagent for enhancing chemiluminescence of 1,2-dioxetane compounds and a method for using the enhancing reagent to enhance the chemiluminescence are provided, in which the enhancing reagent contains an alkyl bis-quaternary ammonium salt of Formula I. A chemiluminescent composition with a 1,2-dioxetane compound as a substrate and a kit thereof are further provided, which contain a 1,2-dioxetane compound and an alkyl bis-quaternary ammonium salt of Formula I.