A novel electrochemiluminescence (ECL) sensor based on tris(2,2′-bipyridyl) ruthenium(II) (Ru(bpy)3 2+)/electrospun carbon nanofiber (CNF)/Nafion composite films was demonstrated for the determination of atropine. The voltammetric and ECL behaviors of the presented sensor were investigated. The results indicated that the addition of CNF in the composite films could increase the current and ECL intensity of Ru(bpy)3 2+, which were subjected to diffusion-controlled processes. Under optimal experimental conditions, the proposed ECL sensor exhibited a wide linear range (r = 0.9967) of 1 × 10−7–1 × 10−4 M with a detection limit (S/N = 3) of 1 × 10−7 M for atropine. The relative standard deviation for 1 × 10−5 M atropine is 2.86% (n = 7), and the present ECL sensor displayed outstanding stability. The ECL sensor was also utilized for the determination of atropine in urine samples, and the recovery was in the range of 81%–88%.