A water-soluble 1,8-naphthalimide-based fluorescent probe 1, bearing two acetic carboxylic groups, exhibited high selectivity and sensitivity for recognition of casein in water based on aggregation induced synchronous emission (AISE) character. A new fluorometric method to detect casein content in PBS (pH 7.4) was developed by synchronous fluorescence spectra with wavelength intervals of 15 and 60 nm. The linear ranges of the calibration curves were 0.1–4.3 μg mL⁻¹ (Δλ = 15 nm) and 0.1–10.1 μg mL⁻¹ (Δλ = 60 nm) for casein, with a correlation coefficient of 0.9962 and 0.9971. The detection limits were estimated to be 5.6 and 7.5 ng mL⁻¹. The proposed method has been applied to the determination of casein in skimmed milk powder samples and the results are in good agreement with those of the Biuret method.