The lateral flow immunosensor (LFI) is a widely used diagnostic tool for biomarker detection; however, its sensitivity is often insufficient for analyzing targets at low concentrations. Here, an electrochemiluminescent LFI (ECL‐LFI) is developed for highly sensitive detection of troponin I (TnI) using Ru(bpy)₃²⁺‐loaded mesoporous silica nanoparticles (RMSNs). A large amount of Ru(bpy)₃²⁺ is successfully loaded into the mesoporous silica nanoparticles with excellent loading capacity and shows strong ECL signals in reaction to tripropylamine. Antibody‐immobilized RMSNs are applied to detect TnI by fluorescence and ECL analysis after a sandwich immunoassay on the ECL‐LFI strip. The ECL‐LFI enables the highly sensitive detection of TnI‐spiked human serum within 20 min at femtomolar levels (≈0.81 pg mL⁻¹) and with a wide dynamic range (0.001–100 ng mL⁻¹), significantly outperforming conventional fluorescence detection (>3 orders of magnitude). Furthermore, TnI concentrations in 35 clinical serum samples across a low range (0.01–48.31 ng mL⁻¹) are successfully quantified with an excellent linear correlation (R² = 0.9915) using a clinical immunoassay analyzer. These results demonstrate the efficacy of this system as a high‐performance sensing strategy capable of capitalizing on future point‐of‐care testing markets for biomolecule detection.