Ascorbic acid (AA) and alkaline phosphatase (ALP) serve as an important coenzyme and enzyme in multiple biological metabolism reactions, respectively, and abnormal levels of these substrates have been associated with several diseases. Herein, a new and simple fluorescence strategy has been developed for AA and ALP sensing by exploiting CdTe quantum dots (QDs) as an effective signal indicator. This method is mainly based on the selective fluorescence-quenching reaction between Ag+ and CdTe QDs, as opposed to silver nanoparticles (Ag NPs); Ag+ can be reduced to Ag NPs by AA. Furthermore, by taking advantage of AA as a mediator, this strategy is further exploited for ALP assay given that ALP can cause the hydrolysis of l-ascorbic acid-2-phosphate (AAP), which yields AA. Under optimal conditions, controlled generation of Ag NPs and the selective recognition-based sensing system exhibit high sensitivity toward AA and ALP with limits of detection (LODs) of 3 μM and 0.25 U L-1 and linear ranges of detection from 0 to 800 μM and 1 to 1000 U L-1, respectively. Moreover, the sensor was successfully used for assaying AA in fruit juice and ALP in human serum. The results demonstrate that the proposed fluorescence strategy has significant advantages, such as its simplicity, cost-effectiveness, and rapid runtime, and the operational convenience of this label-free method further demonstrates its potential for constructing effective sensors with biochemical and clinical applications.