Metal-organic frameworks (MOFs) are often used as carriers in the preparation of electrochemiluminescent (ECL) materials, and ECL materials stabilized in the aqueous phase can be prepared by encapsulating chromophores inside MOFs by an in situ growth method. In this study, nanocomposites MIL-88B(Fe)-NH@Ru(py) with excellent ECL response were prepared by encapsulating Tris(2,2'-bipyridine)ruthenium dichloride (Ru(py)) inside MIL-88B(Fe)-NH using the one-step hydrothermal method. MIL-88B(Fe)-NH possesses abundant amino groups, which can accelerate the catalytic activation process of KSO, and its abundant pores are also conducive to the enhancement of the transmission rate of co-reactant agents, ions, and electrons, which effectively improves the ECL efficiency. In order to obtain more excellent ECL signals, we prepared aminated biochar (NH-biochar) using Pu-erh tea dregs as precursor and loaded gold nanoparticles (Au NPs) on its surface as substrate material for modified electrodes. Both NH-biochar and Au NPs can also be used as a co-reactant promoter to catalyze the activation process of co-reactant KSO. Therefore, a sandwich-type ECL immunosensor was prepared based on a dual signal-enhanced strategy for the highly sensitive and selective detection of aflatoxin B1 (AFB1). Under the optimal experimental conditions, the sensitive detection of AFB1 was achieved in the range of 1 pg·mL~100 ng·mL with a detection limit of 209 fg·mL. The proposed dual signal-enhanced ECL immunosensor can provide a simple, convenient, and efficient method for the sensitive detection of AFB1 in food and agricultural products.