Acid phosphatase (ACP) is an important biomarker for several diseases, such as prostate cancer, Gaucher disease and kidney disease. Therefore, developing sensitive and highly selective assays for ACP is of great significance. To date, several assays for ACP have been reported, including immunoassay, spectrophotometry, chromatography and electrochemistry. Among these methods, fluorescence-based assays have gained prominence due to their high sensitivity, good selectivity, efficiency, and precision. In this study, a novel “turn-on” fluorescent substrate for ACP, named APBA-PLP (ABPL), was synthesized through the Schiff base reaction between 2-aminophenyl boronic acid (2-APBA) dimer and pyridoxal phosphate (PLP). After the reaction, the characteristic FTIR peaks associated with —CHO groups of PLP and —NH2 groups of 2-APBA dimer disappear, and a new FTIR band, originating from CN vibrations of ABPL, appears. In addition, the1H and1H-1H COSY NMR spectra display all the ABPL signals. The FTIR and NMR results indicate the successful synthesis of ABPL. Furthermore, ABPL is applied to the detection of ACP. We observe an increase in fluorescence intensity of the ABPL solution upon adding ACP. This phenomenon is attributed to the aggregation-induced enhanced emission property of the 2-APBA dimer, which arises from its highly ordered stacking. The highly ordered stacking structure is disrupted upon the introduction of PLP onto the 2-APBA dimer (i.e., the synthesis of ABPL). Subsequent addition of ACP leads to the hydrolysis of PLP into pyridoxal, causing the detachment of PLP from the 2-APBA dimer molecule. The 2-APBA dimer molecules can then reassemble into a highly ordered structure, increasing the fluorescence intensity. The relative fluorescence intensity at 376.5 nm exhibits an excellent linear relationship (R2=0.99) with ACP activity in the 0～5 U·L-1 range. Moreover, when ACP, pectinase, papain, and lipase activity were 4, 50 000, 80 000, and 10 000 U·L-1, respectively, the corresponding relative fluorescence intensities were 0.2, -0.006, 0.03, and 0.05. This result confirms the high selectivity of ABPL towards ACP. ABPL can be easily synthesized and exhibits a linear and highly selective response to ACP, presenting a new strategy for the design and synthesis of efficient fluorescent substrates for ACP detection.