Electrochemiluminescence (ECL) biosensors are an ideal choice for the real-time and sensitive detection of biomarkers. However, there are many interfering biomolecules in human serum or cell lysate that can affect the ECL signal through nonspecific interface adsorption. To solve this issue, an efficient ECL antifouling biosensor based on a red blood cell membrane (RBCM) hydrogel was developed for the direct detection of alkaline phosphatase (ALP) in human serum and cell lysate. Among them, the RBCM hydrogel was prepared by embedding the purified RBCM vesicles into [1,2-distearoyl-