The development of rapid, convenient and reliable assays for monitoring alkaline phosphatase (ALP) levels is valuable for clinical diagnoses and biomedical research. In the current study, a ratiometric assay of ALP activity has been realized by covalent immobilization of fluorescein onto polyethylene terephthalate (PET) fibers, followed by electrostatic adsorption of bisquaternary ammonium salt of tetraphenylethene (TPE-2N(+)). In the absence of ALP, the complex formation between phosphorylated fluorescein and TPE-2N(+) results in the aggregation-induced emission (AIE) of TPE at 471 nm. While in the presence of ALP, the hydrolysis of phosphoesters leads to a gradual removal of TPE-2N(+) and the restoration of fluorescein emission at 514 nm. Fibers with surface amine densities of 30 nmol/mg show the most significant and almost linear increases in I-514/I-471 ratios from 0.73 to 3.05 with increasing ALP concentrations from 0 to 100 mU/mL. The ratiometric fluorescence responses result in color changes of fibrous strips from blue (TPE-2N(+)) to green (fluorescein) under an ultraviolet lamp in a matter of minutes. 'The color changes are more suitable for an eyeball detection of ALP levels ranging from 0 to 80 mU/mL, which is included in the concentration range of ALP in human serum considering the dilution factor if necessary. The ALP detection indicates no apparent interference by serum components and good agreement with enzyme-linked immunosorbent assay (ELM). Thus, this is the first study on ratiometric fluorescent assay of serum ALP levels by fibrous strips, which offers a capacity to exploit electrospun fibrous mats and ratiometric responses for real-time assays of bioactive substances as self-test devices.