Ascorbic acid (AA) and alkaline phosphatase (ALP) in human serum are indispensable substances in the process of biological metabolism, and their abnormal contents are strongly associated with a range of diseases. Therefore, it is meaningful to develop a simple, accurate and sensitive assay for the detection of AA and ALP. Up-conversion nanoparticles (UCNPs) have some unique characteristics, especially, the potential for background-free excitation by the near-infrared region (NIR) significantly improves the limits of detection (LODs). Moreover, sensors based on the inner filter effect (IFE) do not require covalent linkage between receptors and fluorophores or surface modification, thus the probe fabrication is simple and flexible UCNP-based. Therefore, a simple, selective, sensitive, and flexible sensing system for the detection of AA and ALP is proposed by combining the IFE and the cyclic signal amplification strategy. Under ideal circumstances, the LODs of ALP through the colorimetric and fluorescence method were 0.192 U/L and 0.013 U/L. The established sensing platform was applied to assessing ALP activity in human serum, and the detection results are close to those of commercially available kits, indicating its immense potential as an effective and convenient tool for monitoring ALP activity in clinical settings.