A 4-aminonaphthalimide-based ratiometric fluorescent probe 1 employing the internal charge transfer (ICT) mechanism was designed and synthesized to detect bovine serum albumin (BSA). The interaction of 1 and BSA was investigated by fluorescence and UV-vis absorption spectroscopy. Upon treatment with BSA, the probe successfully exhibited a ratiometric fluorescent response at 540 nm and 480 nm. The fluorescent intensity ratio at 540 nm and 480 nm (F₅₄₀/F₄₈₀) increases linearly with BSA concentration in the range of 0–75.0 μg mL⁻¹ and the detection limit was about 2.4 ng mL⁻¹. Our strategy is expected to provide a methodology to quantify BSA either by a normal or by a ratiometric and colorimetric way with high sensitivity.