Simultaneous anodic and cathodic electrochemiluminescence (ECL) emissions of black phosphorus nanosheets (BPNSs) and graphitic carbon nitrides (g-C3N4) were reported based on the co-existence of different co-reactants. Anodic ECL was obtained at the BPNSs modified electrode with tripropylamine (TPrA) as a co-reactant, while g-C3N4 was selected as another emitter to obtain cathodic ECL emission with K2S2O8 as co-reactant. Employing the superiority of two separate ECL systems, a facile ECL method was developed for cholesterol detection based on cholesterol oxidase (ChOx) immobilized g-C3N4/BPNSs modified glassy carbon electrode (g-C3N4/BPNSs/GCE). False positive signals can be significantly reduced based on the separation of anode and cathode ECL signals from BPNSs and g-C3N4, respectively. The proposed biosensor provided a quantitative readout proportional to cholesterol concentrations in the range from 0.5 μM to 0.5 mM with a detection limit of 0.14 μM (cathodic system, 3σ, n = 6) and 0.32 μM (anodic system, 3σ, n = 6). The proposed biosensor demonstrated excellent analytical performance with remarkable sensitivity, manifesting its potential application in enzymatic biosensing field.