A sensitive sensing method was developed for the determination of alkaline phosphatase (ALP) activity based on gold nanocluster (Au NC)-catalyzed luminol–H₂O₂ chemiluminescent (CL) reaction. The CL signal of luminol–H₂O₂–Au NCs can be quenched by ascorbic acid, which was the product of magnesium ascorbyl phosphate (MAP) hydrolysis reaction catalyzed by ALP. The proposed sensing platform showed convenient, sensitive and selective detection of ALP in the range of 0.0027 – 1.3890 U L⁻¹, with the detection limit of 0.0026 U L⁻¹. The broad detection linear range and ultra-high sensitivity were inherited from the efficient free radical scavenging capability of ascorbic acid on the luminol–H₂O₂–Au NCs CL reaction. The CL sensing platform was applied to the detection of ALP activity in serum samples. We believe that this sensing platform is a universal CL strategy for ALP detection because ascorbic acid is an efficient CL quencher for many CL reactions.