Autophagy plays a vital role in maintaining intracellular homeostasis through a lysosome-dependent intracellular degradation pathway, which is closely related to the polarity and ATP. Herein, the first example of the dual-response fluorescent probe Lyso-NRB was reported for visualizing the fluctuation of polarity and ATP in lysosomes during autophagy. Probe Lyso-NRB is non-fluorescent. After the decrease of polarity, Lyso-NRB exhibits significant green emission due to the unique intramolecular charge transfer (ICT) effect. Upon the addition of ATP, the probe can react with ATP to rapidly open the spirocycle of rhodamine and a strong red emission can be observed. Moreover, Lyso-NRB exhibits a high sensitivity and selectivity toward polarity and ATP. Most importantly, the probe possesses a good lysosome-targeting ability and is used for the real-time monitoring of lysosome polarity and ATP fluctuations during H2O2 or starvation induced autophagy in living cells. Interestingly, it is found that that ATP deficiency can induce autophagy to increase lysosome polarity. Furthermore, the probe is applied for imaging the change of polarity and ATP under oxidative stress induced autophagy in zebrafish. Therefore, this work holds great potential for tracking the autophagy procedure by detecting the changes of lysosome polarity and ATP, which makes it a potentially powerful tool for understanding the roles of autophagy in diverse biological processes.