Alkaline phosphatase (ALP) is an indispensable hydrolase in living organisms and the abnormality of ALP activity is correlated with a variety of diseases. Exploring ALP activity is important for clinical diagnosis and biomedical research to understand its physiological function. In this study, a dual-mode biosensing platform was constructed based on Cu-based metal-organic frameworks (Cu-MOFs) for electron spin resonance (ESR) and ultraviolet-visible (UV-vis) sensing of ALP. Cu-MOFs, as peroxidase mimics, catalyzed the decomposition of hydrogen peroxide (H2O2) and the generation of reactive oxygen species (ROS) which could oxidize ABTS into ABTS˙+ with good ESR and UV-vis signals. Pyrophosphate ions (PPi) with high affinity to Cu2+ in Cu-MOFs could suppress the peroxidase-like activity of Cu-MOFs, and ALP could hydrolyze PPi, resulting in the recovery of Cu-MOF catalytic activity. Thus, a quantitative dual-mode method for detection of ALP activity was established with good linearity in the range of 0-42 U L-1 and limits of detection as low as 0.386 and 0.523 U L-1 respectively for ESR and UV-vis detection. Benefiting from its high sensitivity and excellent selectivity, this method was applied for ALP detection in human serum and satisfactory recoveries were achieved. The off-on dual-mode sensing platform is more reliable than the single-mode sensor and shows merits like simple operation and cost-friendliness, making it have great potential in the diagnosis of ALP-related diseases.