Herein, a simple, facile, and label-free electrochemiluminescence (ECL) aptasensor platform was constructed by integration of aptamer-gated systems and vertically ordered mesoporous silica films (MSFs) grown in suit of indium-tin oxide (ITO) electrode. In this aptasensor platform, aptamer could be effectively adsorbed on the surface of aminated MSFs by noncovalent electrostatic attraction and then employed as an ideal gate material to control the blocking and releasing of luminescence reagents (Ru(bipy)32+) entrapped within the pores of MSFs. In the presence of target, the specific aptamer-target binding could trigger the uncapping the pores, releasing the Ru(bipy)32+ with detectable reduced of ECL signal. The feasibility and universality of this design was validated by employing three aptamers that bind to lysozyme, adenosine, and K+ as gate materials, and the detection limits were determined to be 0.06 nM, 0.75 nM, and 0.5 μM, respectively. This ECL aptasensor, based on the simple competitive procedure, was simple design, undemanding, and fast in operation. In addition, no other chemical modification of the aptamer was required, suggesting that this ECL aptasensor could be applied to many other target detections just by altering the aptamer sequence.