A novel fluorescent probe named NJQ was synthesized from rhodamine 6 G, and its structure was characterized by Nuclear magnetic resonance spectroscopy, infrared spectroscopy, and mass spectrometry. The probe showed good selectivity for Al3+ and formed a fluorescent system with strong anti-interference ability. The fluorescence intensity of the probe solution was positively correlated with the concentration of Al3+, with an LOD of 1.08 × 10−8 mol/L, with good accuracy and precision. The two are reversibly combined in a 1:1 way. The optimal time for the reaction was 5 min, and the optimal pH was 7. It speculated the possible detection mechanism. In addition, the NJQ probe was successfully applied to determine Al3+ in tap water, and cytotoxicity and fluorescence imaging tests were performed. The results showed that the probe has potential practical applications.